Liver Biopsy

LIVER BIOPSY
INDICATIONS,
PROCEDURES, RESULTS
Edited by Nobumi Tagaya
LIVER BIOPSY INDICATIONS,

PROCEDURES, RESULTS
Liver Biopsy - Indications, Procedures, Results

http://dx.doi.org/10.5772/46113
Contributors
Firpi, Alpna Limaye, Lisa Dixon, Ludmila Viksna, Ilze Strumfa, Janis Vilmanis, Andrejs Vanags, Zane Simtniece, Dzeina
Sulte, Arnis Abolins, Janis Gardovskis, Ervins Vasko, Javier Crespo, Joaqun Cabezas, Marta Mayorga, Hiroyasu
Morikawa, Jean-Franois Cadranel, Jean-Baptiste Nousbaum, Ivan Tokin, Anna Mania, Pawel Kemnitz, Magdalena
Figlerowicz, Wojciech Sluzewski, Aldona Woniak, Nobumi Tagaya, Nana Makino, Masatoshi Oya, Yoshitake
Sugamata, Claudia Randazzo, Anna Licata, Piero Luigi Almasio, Teresa Casanovas, Costin Teodor Teodor Streba, Letitia
Adela Maria Streba, Daniela Dumitrescu, Eugen Florin Georgescu, Monica Lupsor, Horia Stefanescu, Diana Feier, Radu
Ion Badea
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Liver Biopsy - Indications, Procedures, Results, Edited by Nobumi Tagaya

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Contents
Preface VII
Section 1
An Overview and Future Aspects of Liver Biopsy
Chapter 1
Liver Biopsy - Indications, Procedures, Results 3

Claudia Randazzo, Anna Licata and Piero Luigi Almasio
Chapter 2
Types of Liver Biopsy 23

Nobumi Tagaya, Nana Makino, Kazuyuki Saito, Takashi Okuyama,
Yoshitake Sugamata and Masatoshi Oya
Chapter 3
Rethinking the Role of Liver Biopsy in the Era

of Personalized Medicine 33
Teresa Casanovas Taltavull
Section 2
Clinical Practice of Liver Biopsy 73
Chapter 4
Safety and Reliability Percutaneous Liver Biopsy Procedure in

Children with Chronic Liver Diseases 75
Anna Mania, Pawe Kemnitz, Magdalena Figlerowicz, Aldona
Woniak and Wojciech Suewski
Chapter 5
Risks and Benefits of Liver Biopsy in Focal Liver Disease 85

Letiia Adela Maria Streba, Eugen Florin Georgescu and Costin
Teodor Streba
Chapter 6
Current Trends in Liver Biopsy Indications for

Chronic Liver Diseases 103
Jean-Franois Cadranel and Jean-Baptiste Nousbaum
VI
Contents
Chapter 7
Primary and Metastatic Tumours of the Liver: Expanding Scope

of Morphological and Immunohistochemical Details
in the Biopsy 115
Ilze Strumfa, Janis Vilmanis, Andrejs Vanags, Ervins Vasko, Dzeina
Sulte, Zane Simtniece, Arnis Abolins and Janis Gardovskis
Chapter 8
Nonalcoholic Fatty Liver Disease: A Pathological View 161

Joaqun Cabezas, Marta Mayorga and Javier Crespo
Chapter 9
Liver Biopsy After Liver Transplantation 189

Alpna R. Limaye, Lisa R. Dixon and Roberto J. Firpi
Section 3
Non-Invasive Alternatives of Liver Biopsy 207
Chapter 10
Non-Invasive Evaluation of Liver Steatosis, Fibrosis and

Cirrhosis in Hepatitis C Virus Infected Patients Using
Unidimensional Transient Elastography (Fibroscan) 209
Monica Lupsor, Horia Stefanescu, Diana Feier and Radu Badea
Chapter 11
Computer Image Analysis of Liver Biopsy Specimens in Patients

with Heroin Abuse and
Coinfection (Tuberculosis, HCV, HIV) 235
Ivan B. Tokin, Ivan I. Tokin and Galina F. Filimonova
Chapter 12
Future Aspects of Liver Biopsy: From Reality to Mathematical

Basis of Virtual Microscopy 257
Ludmila Viksna, Ilze Strumfa, Boriss Strumfs, Valda Zalcmane,
Andrejs Ivanovs and Valentina Sondore
Chapter 13
Real-Time Tissue Elastography and Transient Elastography for

Evaluation of Hepatic Fibrosis 281
Hiroyasu Morikawa
Preface
Liver biopsy is a procedure that involves obtaining a small piece of liver tissue, which is
then analyzed in the laboratory. Liver biopsy may be used to evaluate a mass seen on
ultrasound, CT or MRI images, diagnose unexplained liver diseases or abnormal liver
function tests, determine the severity of the liver diseases including non-alcoholic liver
disease, certain liver disease such as chronic hepatitis B or C, primary biliary cirrhosis,
primary sclerosing cholangitis, autoimmune hepatitis, hemochromatosis or Wilsons
disease, and monitor the liver after a liver transplantation. Liver biopsy is a safe procedure
when performed by an experienced doctor. However, it is an invasive procedure, and the
common problems include mild pain and a decrease in blood pressure. Although more
serious complications such as bleeding, infection, and injury of nearby organs are rare but
potentially lethal, the monitoring within 24 hours after the liver biopsy is important. This
book describes the role, indication, contraindication, technique and evaluation of outcome of
liver biopsy. I believe that it will be greatly useful to the readers. Furthermore, this book
introduces transgastric liver biopsy using NOTES technique and non-invasive alternatives
including elastography and computer analysis of liver fibrosis as new tools for the
evaluation of liver diseases.
Nobumi Tagaya, M.D., Ph.D.
Department of Surgery,
Dokkyo Medical University Koshigaya Hospital,
Koshigaya, Saitama,
Japan
Section 1
An Overview and Future Aspects of Liver Biopsy
Chapter 1
Liver Biopsy Indications, Procedures, Results

Claudia Randazzo, Anna Licata and
Piero Luigi Almasio
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/52616
1. Introduction
Liver biopsy (LB) is the most common procedure performed in clinical hepatology. His
tological assessment of the liver, and thus, LB is traditionally the reference standard
in the diagnosis and management of parenchymal liver diseases. Definitive diagnosis of
ten depends on LB, and much of understanding of the characteristic features and natu
ral history of liver diseases is based on information obtained by serial liver biopsies.
During the last 60 years as the result of a better understanding of liver disorders, ap
pearance of newer entities and advent of novel hepatic imaging techniques, the indica
tions for LB have evolved. Whereas in the past LB was often performed as the initial
investigation in the workup of liver disease of unknown aetiology, today the most com
mon indication for LB includes staging of chronic hepatitis. A variety of methods exist
for getting a liver tissue specimen. These take account of a percutaneous method, a
transvenous (transjugular or transfemoral) approach, and intra-abdominal biopsy (laparo
scopic or laparotomic). All LB techniques require specific training so as to ensure appro
priate-sized specimen retrieval and the lowest rate of complications. However, because
LB is an invasive procedure that carries a definite, albeit small, risk of complications,
controversy persists with regard to its precise indications in various clinical situations,
its clear contraindications, the optimal technique for its performance (and whether cer
tain modifications improve its safety), and training requirements for clinicians. The aim
of this chapter will be summarize the existing clinical practice of LB with an emphasis
on the technique, indications, contraindications, quality of LB specimens and risk of
complications.
2. Indications
Historically, LB was applied almost exclusively as a diagnostic tool [1]. Nevertheless, as the
result of natural history data and the introduction of many new therapies for patients with
liver disease, histological assessment of the liver has now got on an important role in clinical
management. Therefore, LB currently has three major indications: for diagnosis, for assess
ment of prognosis and/or to assist in the management of patient with known liver disease.
Diagnosis
Identification and staging of parenchymalandcholestatic liver diseases
-alcoholic liver disease
-non-alcoholic steatohepatitis
-autoimmune hepatitis
-primary biliary cirrhosis
-primary sclerosing cholangitis
-metabolic and mitochondrial storage liver diseases such as Wilsons disease, hemochromatosis, Gauchers disease
Evaluation of persistent abnormal liver biochemical tests after negative or inconclusive serologic workup
Evaluation of the type and extent of drug-induced liver injury
Evaluation of fever of unknown origin or immunocompromised patients with hepatomegaly or elevated liver
enzymes levels
Diagnosis of multisystem infiltrative disorders
- Identification and determination of the nature of focal/ diffuse intrahepatic abnormalities on imaging studies
Prognosis - Staging of known liver disease
Evaluation of pre-transplant living-related donor
Evaluation of post-transplant patient with abnormal liver tests (rejection vs. infectious aetiology)
Management Developing treatment plans based on histologic analysis
Pre-treatment evaluation and staging of chronic hepatitis
Evaluation of effectiveness of therapies for liver diseases (eg, autoimmune hepatitis)
Table 1. Indications for liver biopsy
LB is performed to evaluate diffuse parenchymal or focal liver disease (see table 1). LB is
mainly helpful in patients with diagnostic uncertainty(eg, in patients with atypical features).
Available data show that liver histology will, in a proportion of patients, point to a specific
diagnosis [2] and lead to a change in patient management [3,4]. LB has long been considered
as an important diagnostic adjunct in the evaluation of otherwise unexplained abnormalities
of liver biochemical tests. For example, LB may exclude serious liver disease or detect un
suspected non-alcoholic fatty liver disease (NAFLD) or intrahepatic sclerosing cholangitis
after an otherwise negative biochemical, serologic and radiologic evaluation [3]. Needle LB
for diagnosis remains important in cases of coexisting disorders such as steatosis and HCV
[5] or an overlap syndrome of primary biliary cirrhosis (PBC) with autoimmune hepatitis
(AIH) [6].
Liver Biopsy: An Overview

http://dx.doi.org/10.5772/52616
Other indications for LB include documentation of alcoholic liver disease and assessment of
its severity; evaluation of otherwise unexplained fever, particularly in patients with Ac
quired Immune Deficiency Syndrome (AIDS); detection of underlying granulomatous liver
disease. LB also provides important diagnostic information regarding drug-induced liver in
jury. Liver histology is appropriately considered in conjunction with clinical and laboratory
data in case of hereditary disorders, eg hemochromatosis (quantitation of the level of iron),
Wilsons disease (quantitation of the level of copper), and alpha-1 antitrypsin deficiency.
Liver histology may also be useful in detection of infiltrative processes such as amyloidosis
[7]. Moreover, liver histology is often helpful in the setting of acute liver failure (ALF) [8].
An additional main use of LB is in assessing disease severity, particularly fibrosis, which, as
a precursor to cirrhosis, may predict the emergence of complications of portal hypertension
and also liver-related morbidity and mortality.
Owing to the wide use and superior resolution of cross-sectional imaging such as ultraso
nography (US), computed tomography, and magnetic resonance imaging, focal lesions are
being detected more often. Fortunately, the same technologic advances allow us to confi
dently establish a diagnosis without biopsy in most cases. Nevertheless, sometimes a biopsy
of a suspected neoplasm will help change management. In this case, careful consideration of
biopsy technique is important, as neoplasms have a higher bleeding risk and the potential to
seed other sites along the biopsy tract or in the abdominal cavity [9]. At present, most biop
sies currently performed for parenchymal disease are not to make a specific diagnosis but to
assess liver damage, particularly in situations where (prognostic) information about fibrosis
may guide consequent treatment. For example, histological analysis of the liver in patients
with chronic HCV-induced liver disease gives information about the grading (inflammatory
activity) and the staging (degree of fibrosis) that predict the course of disease; the treatment
is often advocated for those with at least moderate to severe staging, but may be withheld
when fibrosis is minimal or absent [10]. Liver histology is also generally used in disease
monitoring of patients with AIH [11]. Monitoring the plasma cell score on LB may help pre
dict relapse when a physician is considering reducing or discontinuing immunosuppressive
therapy [12]. For further information on the role of histological analysis in the management
of individual liver diseases, is possible to see guidelines for HCV [10], HBV [13], hemochro
matosis [14], cholestatic liver diseases [15], AIH [11], and Wilsons disease [16].
Assessment of liver histology after orthotopic liver transplantation (OLT) is highly valuable
to assess for allograft rejection and the presence and intensity of disease recurrence. Contro
versy persists regarding the precise indications for LB. Among these controversies are the
following:
The precise cut-off of serum aminotransferase levels that should prompt a LB: any persis
tent elevation, 1.5 times the upper normal limit, or 2 standard deviations above the mean
[17,18]. Even the definition of the upper limit of normal is controversial [19-21].
The need for LB in patients presumed to have NAFLD. Whereas imaging studies are sen
sitive for detecting steatosis, they are relatively not sensitive and nonspecific for detecting
inflammation and fibrosis. Only on liver histology can distinguish fatty liver from steato
hepatitis, which can lead to fibrosis and cirrhosis. LB is often considered if serum alanine
aminotransferase (ALT) levels remain elevated after a modification of lifestyle and risk
factors [22].
The need for LB in all patients with PBC and primary sclerosing cholangitis (PSC). In
most cases the diagnosis can be established on the basis of a cholestatic pattern of liver
chemistries and either anti-mitochondrial antibodies in PBC [6] or endoscopic retrograde
cholangiopancreatography (ERCP) in PSC [23]; scoring systems based on quickly estab
lished clinical variables could be used to assess prognosis and response to therapy.
The need for protocol liver biopsies in all liver transplant recipients. A high rate of histo
logic abnormalities in the absence of liver biochemical test abnormalities has been descri
bed as late as 10 years after transplantation [24].
Overall, in patients without a definitive pre-biopsy diagnosis, LB has been shown to change
the clinical diagnosis in 8% to 10% and to change the management in 12% of patients [25].
However, changes in management are often of minor importance [3].
3. Biopsy technique
Performance of LB requires an adequate sized and dedicated space suitable for focused
physician effort as well as safe patient recovery. There are different approaches for ob
taining liver tissue: percutaneous, transjugular, laparoscopic, and intraoperative, each
having advantages and disadvantages. The biopsy technique is chosen on the basis of
the indication, risks, and benefits in the individual patient. The most common approach
for collecting a liver sample is percutaneous LB, either blinded or under US guidance. It
is quick and safe procedures commonly performed by gastroenterologists or hepatolo
gists in out-patient settings.
A variety of needles are available for percutaneous LB; they are broadly classified into suc
tion needles (Menghini, Klatskin, Jamshidi), cutting needles (Vim-Silverman, Tru-cut), and
spring-loaded cutting needles that have a triggering mechanism. The choice of a specific
type of needle depends in part on local preference. Cutting needles usually produce a larger
sample and are less likely to yield inadequate specimens than are suction needles, but they
probably result in more complications [26], probably because the needle remains in the liver
longer. Cutting needles can be useful in patients with cirrhosis. Suction needles are quicker
(in the liver for a briefer time), easier to use, and less expensive, but tend to produce more
fragmented samples. Disposable biopsy needles and biopsy guns are often used. A typical
biopsy gun uses a modified 18-, 16-, or 14-gauge Tru-cut needle that is fired by a fast and
powerful spring mechanism.
If the patient is not relaxed, a mild sedative should be administered just before the biopsy
[27]. The current data on the use of prophylactic antibiotics is inconclusive. Prophylactic an
tibiotics have been recommended for patients at increased risk of endocarditis or with bili
ary sepsis [28]. However, recent results suggest that prophylactic administration of

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antibiotics following apercutaneous liver biopsy does not have a significant impact on the
post-procedure results or incidence of infection [29]. During the procedure, patients placed
in the supine position with the right hand resting behind the head [30]. For the blind ap
proach (also referred to as the percussion-palpation approach), caudal percussion is helpful
in selecting the site for the biopsy over the hemithorax between the anterior and mid-axil
lary lines, until an intercostal space is reached where dullness is maximal at the end of expi
ration. The intercostal space below this point (usually in the 7th-8th intercostal space) is used.
A local anesthetic, typically lidocaine (without adrenaline), is administered with a 25-gauge
needle first subcutaneously and into the intercostal muscle and finally down to the dia
phragm and the capsule of the liver to reduce pain. The biopsy is performed while the pa
tient holds a breath in full expiration [31]. With a suction needle, aspiration is applied, and
the needle is rapidly introduced perpendicularly to the skin into the liver and withdrawn
quickly (within 1 second). This is the critical step in performing the biopsy to minimize the
risk of lacerating the liver and inducing bleeding. If insufficient tissue is obtained on the first
pass [32], a second pass is performed at a different angle. After the biopsy, the patients is
usually kept on the right lateral decubitus position for up to 2 hours to reduce the risk of
bleeding and the pulse and blood pressure are monitored. Post-procedure monitoring has
evolved over time. Most complications manifest within the first few hours [26], and under
certain circumstances more and more patients are being discharged just 1 or 2 hours after
imaging-guided biopsy. Rightly, the recommended observation time after biopsy is between
2 to 4 hours. To direct the needle away from other organs and large vascular structures,
physicians often use US guidance. The US has been used either throughout the entire proce
dure (real-time) or immediately before (site marking) through a technique in which the pa
tient subsequently has LB performed at the marked site. US guidance is the most
controversial issue associated with LB [33-35]. Potential LB sites marked by percussion were
changed in between 3 and 15% of patients after US was performed [36,37]. In an uncontrol
led Italian study, routine identification of the puncture site by US led to a diagnostic tissue
sample in 99% of patients [35]. In diffuse liver disease, US marking or guidance has been
associated with lower rates of pain, hypotension, and bleeding [31]. In a survey of 2084 liver
biopsies in France, US guidance is used in 56% of cases (in 34% to determine the puncture
site and in 22% to guide the biopsy) and is thought to reduce the frequency of severe com
plications [38]. Cost-effectiveness analyses have suggested that routine US guidance in clini
cal practice increases the cost of LB but may be cost-effective, with an incremental cost of
$2731 to avoid one major complication [39,40]. In addition, a large, randomized, prospective
trial found that US use lowered the rate of post-biopsy hospitalization (most common rea
son for hospital admission was pain). Indeed there is a long track record of safety for per
forming percutaneous LB without imaging guidance. Thus, the role of US to guide
percutaneous LB remains controversial. Use of ultrasound is not mandatory. A transjugular
biopsy route offers a reasonable alternative to standard biopsy in high-risk patients (eg pres
ence of massive ascites, severe coagulopathy, morbid obesity with a difficult to identify
flank site or fulminant hepatic failure) [41]. With transjugular LB, the liver tissue is obtained
from within the vascular system, which minimizes the risk of bleeding [42,43]. The proce
dure is performed by interventional radiologists or hepatologists under X-ray videofluoro
scopy. Electrocardiographic monitoring is required to detect arrhythmias induced by

passage of the catheter through the heart [41,44]. The patient is positioned supinely, with the
head rotated opposite to that of the right internal jugular vein to be punctured, under local
anesthesia using the Seldinger technique; then, a catheter is introduced into the hepatic vein
under fluoroscopic control, and a needle biopsy of the liver performed through the catheter.
Samples are retrieved from a Menghini or Tru-cut needle passed through the catheter into
the liver. The transjugular approach permits concomitantly measurement of hepatic venous
pressure gradient or opacification and imaging of the hepatic veins and inferior vena cava
[45] helping in the diagnosis and management of select group of patients, particularly those
with cirrhosis. In the past, a drawback of transjugular biopsy was the small and fragmented
samples obtained. Better needles and more experience have led to improved quality of
specimens. However, a transjugular LB is available only at a limited number of tertiary care
facilities. Mortality is low (0.09%) [41], but perforation of the liver capsule can be fatal [46].
With laparoscopic approach, specific lesions can be identified and targeted precisely; thus it
is especially useful in the diagnosis of peritoneal disease, the evaluation of ascites of un
known origin and abdominal mass, the staging of abdominal cancer. Laparoscopic LB is a
safe procedure that can be performed under local anesthesia with conscious sedation, al
though it requires expertise that is not readily available. Absolute contraindications include
severe cardiopulmonary failure, intestinal obstruction, bacterial peritonitis; relative contra
indications are severe coagulopathy, morbid obesity, and a large ventral hernia [33]. For
most parenchymal liver diseases, the extra time and cost required for laparoscopy are not
justified by the increased yield. Liver biopsies (needle or wedge) can also be obtained dur
ing abdominal surgery whenever liver disease is suspected. In many instances, an abnormal
appearance of the liver during surgery for an unrelated procedure (most often cholecystec
tomy) is the first indication of an underlying liver disease. It is generally performed either
with typical needle devices or by wedge resection by those with special expertise. While in
traoperatively obtained liver biopsies have the added advantage of obtaining adequate tis
sue sampling under direct vision from grossly visible/suspicious lesions, they are
suboptimal for assessment of liver fibrosis and inflammation, due to preponderance of Glis
sens capsule, wider portal tracts in the subcapsular area, and frequent but inconsequential
surgically induced hepatitis. Other advantages are the ability to evaluate for potential extra
hepatic spread of malignancy and to look for a cause of unexplained ascites (peritoneal bi
opsy). The major disadvantages are cost and the added risk of anesthesia. Therefore, needle
biopsy should be the technique of choice at laparotomy.
4. Contraindications
Although LB is often essential in the management of patients with liver disease, physicians
and patients may find it to be a difficult undertaking because of the associated risks.
The consensus guidelines of contraindications for percutaneous LB are listed in Table 2.

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Absolute
Uncooperative patient
History of unexplained bleeding
Tendency to bleed
-Prothrombin time "/> 3-4 sec over control
-Platelet counts < 50.000/mm3
-Prolonged bleeding time (10 min)
Unavailability of blood transfusion support
Recent use of aspirin or other nonsteroidal anti-inflammatory drugs (within last 7-10 days)
Relative
Ascites
Morbid obesity
Infection in the right pleural cavity or below the right hemidiphragm
Suspected hemangioma or other vascular tumor
Hydatid disease (Echinococcal cysts)
Table 2. Contraindications to percutaneous LB
Percutaneous LB with or without image guidance is appropriate only in cooperative pa

tients. As for any procedure, the patient that undergoes a LB should be able to understand
and cooperate with the physicians instructions. An academic concern is that if the patient
accidentally moves when the biopsy needle is in the liver, then a tear or laceration may oc
cur (which would in turn greatly increase the risk of bleeding). Thus uncooperative patients
who require LB should undergo the procedure under general anesthesia or via the transve
nous route.
Coagulopathy is generally considered a contraindication to percutaneous LB, but the precise
parameters that preclude LB are unsettled [47]. Generally, LB should be withdrawn when
the prothrombin time (PT) is more than 3-4 seconds above the control value (International
Normalized Ratio, INR>1.5) or when the platelet count is less than 60.000/mm3 [48]. Never
theless, it is important to emphasize that the relationship of abnormal indices of peripheral
coagulation to the occurrence of bleeding after LB in patients with acute as well as chronic
liver disease is uncertain, as limited data are available [47,49]. In patients with mild to mod
erate prolongation of PT, administration of fresh frozen plasma or appropriate clotting fac
tor concentrates may allow safe performance of a LB, as in hemophiliacs [50]. A low platelet
count is probably less likely to result in bleeding in a cirrhotic patient with hypersplenism
than in a leukemic patient with a comparable platelet count but platelet dysfunction. Proba
bly, platelet dysfunction due to aspirin use is a major risk factor as well. Whether patients
with renal insufficiency are at increased risk of bleeding complications after LB is also un
certain [28]. In summary, the decision to perform LB in the setting of abnormal hemostasis
parameters should continue to be reached as the result of local practice because there is no
specific INR and/or platelet count cut-off at or above which potentially adverse bleeding can
be reliably predicted.
10
A LB is precluded by tense ascites, because the liver will bounce away from the needle,
thereby preventing adequate sampling of tissue, and the ascites will provide insufficient
tamponade in case of bleeding. In patients with tense ascites requiring a LB, a transvenous
approach is commonly recommended. Acceptable options include total paracentesis per
formed immediately prior to percutaneous biopsy or transvenous or laparoscopic biopsy.
Relative contraindication is morbid obesity; in this case, transjugular biopsy is a logical al
ternative.
A standard LB is probably contraindicated by extrahepatic biliary obstruction, bacterial
cholangitis, and the risk of bleeding after LB appears to be increased in patients with a
known hematologic malignancy involving the liver [28].
Although LB in patients with mass lesions is usually safe, biopsy of known vascular lesions
(ie hepatic hemangioma) should generally be avoided [51]. Patients who require LB and
who have a large vascular lesion identified on imaging should undergo the procedure using
real-time image guidance. Biopsy of potentially malignant lesions should be undertaken
with care because it is believed that tumour vessels are more likely to bleed [51] and it can
be also associated with a risk of tumour spread [52,53].
Biopsy of infectious lesions is generally safe. In the past, the presence of an echinococcal cyst
was considered a contraindication to LB, because of the possibility of disseminating cysts
throughout the abdomen and the risk of anaphylaxis. However, with recent advances in
treatment, echinococcal cysts can be aspirated safely under ultrasound guidance [54].
5. Complications
When performing a LB, should be aware of multiple potential complications that may occur
after biopsy.At the time that informed consent is obtained, it is reasonable to outline these
complications clearly, warn the patient of the potential pain, and mention in a general state
ment that other complications, albeit rare, can occur.
Although the percutaneous biopsy is invasive, associated complications are rare, occurring
in up to 6%, and 0.04% to 0.11% can be life threatening [33].
The different complication rates were attributed to variation in technique and to differences
in the needles used, as well as differences in the severity of the liver disease and selection
criteria in different centers.
The most common complication after percutaneous LB is pain [55]. Approximately 25% of
patients have pain in the right upper quadrant or right shoulder; the pain is usually dull,
mild and brief. Right upper-quadrant pain does not seems to be related to approach (i.e.
subcostal vs. intercostal) [56]. The mechanism of pain following percutaneous biopsy is most
likely a result of bleeding or possibly bile extravasation from the liver puncture wound,
with subsequent capsular swelling, although the exact mechanism remains uncertain [57].
When present, pain can generally be managed with small amounts of narcotics. A decision

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about when to investigate with imaging and/or to hospitalize the patient for observation
due to pain should be made on a case-by-case basis.
MAJOR
Dearth
Haemorrhage (intraperitoneal, intrahepatic, haemothorax)
Perforation of the gallbladder or of the bowel
Pneumothorax, haemothorax
Biopsy of the right kidney or the pancreas
Intrahepatic arteriovenous fistula
Bile peritonitis
MINOR
Pain (biopsy site, right upper quadrant and right shoulder pain)
Transient hypotension (vasovagal response)
Pneumoperitoneum
Hemobilia
Infection (bacterial sepsis, local abscess)
Intrahepatic and subcapsular hematoma
Table 3. Complications of percutaneous liver biopsy
Transient hypotension, due to vasovagal reaction, can occur, particularly in patients who are
frightened or emotional.
Major complications were defined as life threatening or those that required hospitalization,
prolonged hospitalization or those that resulted in persistent or significant disability. Most
serious complications occur within 24 hours of the procedure, and 60% happen within 2
hours; between 1% and 3% of patients require hospitalization [33].
The most common serious complication is bleeding because of transection of a vascular
structure [26]; bleeding may occur in the absence of pain. Mild bleeding, defined as that suf
ficient to cause pain or reduced blood pressure or tachycardia, but not requiring interven
tion, occurs in about 1/500 biopsies [58]. Severe bleeding is defined clinically by a change in
vital signs with imaging evidence of intraperitoneal bleeding. Such bleeding has been esti
mated to occur in between 1 in 2.500 to 1 in 10.000 biopsies after a percutaneous approach
for diffuse liver disease [59]. Although very rare, clinically significant intraperitonealhemor
rhage is the most serious bleeding complication of percutaneous LB; it usually becomes ap
parent within the first 2-3 hours after the procedure [26]. Free intraperitoneal blood may
result from laceration of the liver capsule caused by deep inspiration during the biopsy or
may be related to a penetrating injury of a branch of the hepatic artery or portal vein. The
likelihood of hemorrhage increased with older age, presence of cirrhosis or liver cancer, and
number of passes ( 3) with the needle during biopsy. The relationship between LB compli
cations and the number of needle passes is well documented [51]. The frequency of compli
cations increased with the number of passes performed at a rate of 26.4%, with one pass vs.
11
12
68% with two or more passes (P< 0.001) [38]. An additional factor in determining the risk of
hemorrhage may be the type of needle used; cutting needles are more likely to result in
hemorrhage than suction needles [26]. Severe bleeding requires hospitalization and is most
often managed expectantly with placement of intravenous catheters, volume resuscitation
by the administration of intravenous fluids and blood transfusion as necessary. If hemody
namic instability persists for a few hours despite the use of aggressive resuscitative meas
ures, angiography with selective embolization of the bleeding artery or surgery (to ligate the
right hepatic artery or resect a section) is required.
Subclinical bleeding leading to intrahepatic or subcapsular hematomas may be noted after
LB even in asymptomatic patients. It is occurs in up to 23% of patients [60] and can be de
tectable by US. Large hematomas may cause pain associated with tachycardia, hypotension,
and a delayed decrease in the hematocrit [33]. Conservative treatment of hematomas is gen
erally sufficient.
After tranvenous biopsy bleeding is extremely rare because of the Glisson capsule is not
breached except as a procedural complication from within the liver [61].
The least common of the hemorrhagic complications is hemobilia, which usually presents
with the classic triad of gastrointestinal bleeding, biliary pain, and jaundice [26] approxi
mately 5 days after the biopsy [62].
Transient bacteremia has been reported in 5.8 to 13.5 percent of patients after LB [63], and
although such bacteremia is generally inconsequential, septicaemia and shock can rarely oc
cur in patients with biliary obstruction and cholangitis.
Biliary peritonitis caused by puncture of the gallbladder is rare (0.00001% frequency) but
can be fatal [64].
Pneumothorax, hemothorax, subcutaneous emphysema, perforation of any of several organs
(lung, colon, and kidney), subphrenic abscess are other complications reported with LB.
Pneumothorax may be self-limited but may require more aggressive intervention depending
on the severity of symptoms. Visceral perforation is usually managed expectantly. In most
situations, observation is all that is required, although surgical intervention may be needed
in the case of gallbladder puncture and persistent bile leak, or in the case of secondary peri
tonitis.
Differences in complication rates, either minor or major, have been reported between the
blind and US-guided LB. The use of US guidance can prevent inadvertent puncture of other
organs or large intrahepatic vessels. US may also reduce the incidence of major complica
tions such as haemorrhage, bile peritonitis, pneumothorax, etc.
With respect to the impact of the experience of the operator to the rate of complications, the
evidences are controversial. A survey performed in Switzerland showed that the complica
tion rate of percutaneous LB was mainly related to the experience and training of the opera
tor, in particular a lower complication rate was reported for physicians who performed
more than 50 biopsies a year [65]. Another study showed that the rate of complications in
percutaneous LB was 3.2% if the operator had performed <20 biopsies, and only 1.1% if the

http://dx.doi.org/10.5772/52616
operator had performed more than 100 biopsies [64] In contrast, Chevallier et al. showed
that the operators experience did not influence either the final histological diagnosis or the
degree of pain suffered by patients [66].
In adult series, the rate of major complications associated with transjugular LB is low (0.5%;
liver puncture-related, 0.2%; non-liver puncturerelated,0.3%), considering that it is currently
performed in patients with coagulopathy [41]. Minor complications were significantly more
frequent with Menghini needle, possibly related with the difficulty in controlling the depth
of puncture increasing the risk of capsular penetration [46].
MINOR
Pyrexia
Hypotension
Neck hematoma, bleeding
Abdominal pain
Neck pain
Subclinical capsular perforation
Carotide puncture
Small hepatic hematoma
Transient Horners syndrome
Hepatic-portal vein fistula
Transient dysphonia
Hepatic artery aneurysm
Arm numbness/palsy
Biliary fistula
Supraventricular arrhythmia
Haemobilia
MAJOR
Large hepatic hematoma
Ventricular arrythmia
Intraperitoneal haemorrhage
Pneumothorax
Inferior vena cava or renal vein perforation
Respiratory arrest
Table 4. Complications of transjugular liver biopsy
Factors associated with liver and non-liver puncture related complication rates included
number of passes (liver puncture-related), young age, and number of transjugular biopsies.
The complications after laparoscopic LB include perforation of a viscus, bleeding, hemobilia,
laceration of the spleen, leakage of ascitic fluid, hematoma in the abdominal wall, vasovagal
reaction, prolonged abdominal pain, and seizures [67].
The most quoted mortality rate after percutaneous LB is less than or equal to 1/10.000 biop
sies. Mortality is typically related to bleeding. Mortality is highest among patients who un
dergo biopsies of malignant lesions. Cirrhosis is another risk factor for fatal bleeding after
LB. Mortality after transvenous biopsy was 0.09% [41] in adult series, but may reflect the se
lection of higher risk patients for this intervention. Indeed, mortality is significantly higher
in children; smaller livers and horizontal hepatic veins may increase the technical difficulty
and risk of capsular perforation, which might be minimized by combined fluoroscopic and
US guidance [68].
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6. Pathological considerations
Even though LB gives significant diagnostic and prognostic information and helps define
treatment plans, it must be recognized that sampling variability and intra observer variabili
ty may restrain the diagnostic value of LB. The quality of LB is usually determined by
length, width, fragmentation and complete portal tracts (CPTs) [33].
Sample size can affect the diagnostic accuracy of LB specimens [33]. s almost always means
that size of the needle biopsy specimen should be of large enough size to accurately assess
the degree of liver injury. Considering that a biopsy sample taken from an adult corre
sponds to a fraction of just 1/50,000th of the whole liver, a biopsy specimen would seem to
be inadequate in the case of diffuse diseases, such as a chronic viral hepatitis, in which the
liver changes may be unevenly distributed.
Several studies demonstrated that cirrhosis can be missed on a single blind percutaneous LB
in 10%-30% of cases [69-71]. In a detailed study, Colloredo et al. [72] carefully evaluated the
impact of sample size on correct stadiation of liver fibrosis in patients with chronic hepatitis
C. By reducing progressively the dimensions of the same LB, they reported that the smaller
the sample analyzed, the milder the diagnosis made by the pathologist with respect to the
stage of fibrosis. The reduction in length (<2 cm) led to a significant decrease in number of
complete portal tracts and underestimation of grading and staging. The study by Colloredo
et al also introduced the concept of a minimum number of CPTs. Since the number of por
tal tracts is proportional to biopsy size [73], there was evidence that with fewer than 11 to 15
CPTs grade and stage are significantly underestimated [72]. The lower number of complete
portal tracts may explain the lower diagnostic accuracy obtained with smaller samples
[73,74]. Guido and Rugge have suggested that a biopsy sample 20 mm containing at least
11 CPTs should be considered reliable for adequate staging [75]. Other authors have recom
mended even bigger samples, up to 25 mm in length [76]. Scheuer suggested that bigger is
better [77]. Very recently, the American Association for the Study of Liver Diseases
(AASLD) has recommended a biopsy sample of at least 2030 mm in length, and containing
at least 11 CPTs [48].
In summary, an adequate (although probably still imperfect) sample needs to be at least 2
cm long (1.4 mm width, 16G) and to contain no fewer than 11 CPTs. These criteria have been
adopted rapidly as optimal standards.
Of equal importance to adequate specimen size is the necessity that a pathologist experi
enced in liver disease interprets the biopsy, ideally in partnership with the clinician who
performed the biopsy and/or whom is caring for the patient. Rousselet et al. reported
that the degree of experience of the pathologist (specialization, duration, and location of
practice) may have a significant impact on the diagnostic interpretation of LB, even high
er than that related to characteristics of the specimen (length, fibrosis class number, mis
cellaneous factors) [78].
Assessment of disease severity with liver histology is supported by a wide body of liter
ature [79]. Complex scoring systems, such as the Knodell scoring system [80] and its re

http://dx.doi.org/10.5772/52616
vised form, the Ishak scoring system [81] have been developed for grading and staging
of chronic viral hepatitis, and there is now a similar score for steatohepatitis [82]. Never
theless, these are not highly reproducible and are only appropriate for statistical analysis
of (large) cohorts of patients in clinical trials. In clinical practice, it was recommended to
use the simple systems with three to four categories such as METAVIR [83] rather than
complex (Ishak) scoring system [48].
7. Further research
Until a few years ago, LB was the only tool for the diagnosis of liver disease. However, the
indications for performing a LB have undergone changes in the last decade. Given the inva
sive nature of LB, several simple and non-invasive methods (radiologic, immunologic, bio
chemical, genetic markers) have been studied and proposed as surrogates of liver histology.
The main advantages of serum biomarkers vs. LB include being less invasive and the possi
bility to be easily repeated to monitor the status of liver disease. However, at this time, they
are primarily useful for detecting advanced fibrosis or for excluding minimal or no fibrosis.
They are not sufficiently accurate for assessing disease progression or the effect of therapy.
Due to inadequate diagnostic accuracy or to lack of sufficient validation, current guidelines
do not recommend serum biomarkers a substitute for LB that is still considered the refer
ence standard. Notably, non-invasive serum biomarkers, when combined, may reduce by
50%-80% the number of liver biopsies needed for correctly classifying hepatic fibrosis. Se
rum biomarkers for liver fibrosis are particularly useful for the initial assessment as well as
for long-term monitoring of particular subsets of patients (ie, chronic hepatitis C). In this
view, combination algorithms of the most validated non-invasive methods for liver fibrosis
and LB represent a rational approach to the diagnosis of liver fibrosis in chronic liver diseas
es. Novel imaging techniques, such as measuring the elasticity of the liver using transient
elastography (Fibroscan) [84], may assess fibrosis more directly. However, the use of such
techniques in routine clinical practice has not been well defined and require further investi
gation. LB cannot be avoided completely, but should be used in those cases in which noninvasive methods show poor accuracy. Nevertheless, large scale, prospective, independent
studies are needed in other aetiologies of CLDs. Many questions about LB remain and they
require much more research. For instance, it is not clear which biopsy devices or techniques
are best. In addition, few if any studies have assessed the biopsy's long-term effects. Because
the liver is cut and bleeds during procedure, there will be some subsequent scarring.
8. Conclusions
LB continues to play a central role in the evaluation of patients with suspected liver disease,
but many aspects of the procedure remain controversial. For example, the precise degree of
serum ALT elevations that should prompt a LB is debated, as is the need for LB in all pa
tients with suspected NAFLD and chronic hepatitis C. The importance of LB in arriving at a
15
16
diagnosis of diffuse parenchymal liver disease is being diminished by accurate blood testing
strategies for chronic viral hepatitis, autoimmune hepatitis, and primary biliary cirrhosis.
Further, imaging tests are superior to LB in the diagnosis of primary sclerosing cholangitis.
However, many cases remain in which diagnostic confusion exists even after suitable labo
ratory testing and imaging studies. Diagnosing infiltrative disease (eg, amyloidosis, sarcoi
dosis), separating benign fatty liver disease from steatohepatitis, and evaluating liver
parenchyma after liver transplantation are best accomplished by LB.
Percutaneous LB is contraindicated in patients with severe coagulopathy and ascites, but
the degree of coagulopathy that contraindicates a LB is controversial. Also controversial
are the technical aspects of LB, particularly the choice of needle (cutting vs. suction) and
the use of US to mark or guide the biopsy site. Bleeding is the major complication of
LB, with a risk of 0.3%; cutting needles are more likely to cause hemorrhage than are
suction needles. While needle biopsy is still the mainstay in diagnosing hepatic fibrosis,
its days of dominance seem limited as technology improves. When physical examination
or standard laboratory tests reveal clear-cut signs of portal hypertension, LB will seldom
add useful information. Similarly, when imaging studies provide compelling evidence of
cirrhosis and portal hypertension, needle biopsy is not warranted. The combination algo
rithms warrant further evaluation in all chronic liver diseases, as they may help decrease
the number of liver biopsies required. Moreover, transient elastography is playing an ev
er-increasing role in the assessment of hepatic fibrosis and will significantly reduce the
need for biopsy in patients with liver disease.
Clearly, as our knowledge of various liver disorders advances and new especially non-inva
sive diagnostic tests are developed, the role of LB in medical practice will continue to
evolve. Emergence of better imaging techniques, surrogate serological markers of liver fibro
sis are among the many new and exciting developments that hold promise for the future.
Author details
Claudia Randazzo, Anna Licata and Piero Luigi Almasio
Department of Gastroenterology, University of Palermo, Italy
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Chapter 2
Types of Liver Biopsy

Nobumi Tagaya, Nana Makino, Kazuyuki Saito,
Takashi Okuyama, Yoshitake Sugamata and
Masatoshi Oya
http://dx.doi.org/10.5772/52914
1. Introduction
Liver biopsy (LB) is an important procedure in the diagnosis and treatment of liver diseases.
However, procedures for performing LB vary amongst institutions, and no universal guide
lines exist. LB is performed for two main reasons: diagnosis of a liver condition itself, and as
an adjunct to an existing surgical procedure. Recently, it has become possible to employ
both approaches with minimal invasiveness using the transjugular route or under the guid
ance of ultrasound, computed tomography, or laparoscopic and endoscopic ultrasound.
Techniques for liver tissue sampling include percutaneous liver biopsy [1-6], transjugular
liver biopsy [7-14], laparoscopic liver biopsy [15], and transgastric liver biopsy [16-20]. This
chapter introduces these techniques and evaluates their outcomes.
2. Percutaneous Liver Biopsy (PLB)

PLB is performed either blind or under imaging guidance. In the latter context, ultrasound
(US) or computed tomographic (CT) guidance is used. Although these results of US-guided
PLB depend greatly on the skills of the gastroenterologist, hepatologist or radiologist and
the technical capabilities and quality of the US instrument, the available data indicate that it
has a lower complication rate, requires a lower number of passes, is associated with less
pain and pain-related morbidity, has a lower likelihood of the need for a repeat procedure,
affords better-quality tissue specimens, and has only a marginally increased cost in compari
son with blind PLB [21].
24
PLB under image guidance essentially eliminates the risk of pneumothorax, or injury to the
gallbladder or other viscera because the needle track is directly visualize of organ. Pain is
the commonest complication, and up to 75% of patients suffer some discomfort after LB [21].
However, complications after PLB require careful observation. Piccinino et al. [22] reported
that 61% of such complications appeared in the first 2 hours after the biopsy, 82% in the first
10 hours, and 96% in the first 24 hours. Strict observation is therefore required for the first 24
hours after PLB. Several large studies have shown rates of major complication after PLB
ranging from 0.09% to 2.3%, severe complications in 0.57%, and mortality ranging from
0.03% to 0.11% [23-25]. Hardman et al. [4] reported one patient with graft vs. host disease
and hypertension who died after PLB. This patient had multi-organ system failure at the
time of biopsy and died within 24 hours of the biopsy. Furthermore, the complications of
PLB seem to be related to the type of technique employed. In fact, the complications associ
ated with US-guided PLB are significantly lower than those associated with blind PLB: 0.5%
vs. 2.2% for severe complications [26], 2% vs. 4% [27] and 1.8% vs. 7.7% [28] for total compli
cations. PLB under US guidance is recommended as a reasonable and cost-efficient proce
dure [1, 26, 28]. However, EI-Shabrawi et al. [5] have reported that blind PLB performed by
the Menghini aspiration technique is safe even in infants and small children without mortal
ity or major complications such as bile leakage, pneumothorax, and bleeding requiring
blood transfusion. Szymczak et al. [6] also reported the safety and effectiveness of blind PLB
based on an analysis of 1412 procedures, and showed that the rates of complications and
failure were dependent on the experience of the operator. Moreover, the needle used was
the Menghini-type suction needle, which carries a smaller risk of bleeding than cutting nee
dles such as the widely employed Tru-cut needle. They concluded that the risk of complica
tions and failure rate are low if the indications and contraindications are considered
carefully and the biopsy is performed by a skilled and experienced operator.
Furthermore, with regard to bleeding after PLB, Alotaibi et al. [3] have reported that a posi
tive color Doppler sign in US indicates bleeding along the biopsy tract, and that US-guided
compression is effective for achieving appropriate hemostasis. Also, tract-plugging of the bi
opsy tract with Gelfoam or other thrombotic agents, is an important procedure for reducing
the risk of bleeding and subcapsular hematoma in PLB [2]. Nevertheless, in patients with as
cites or abnormal coagulation profiles, another procedure should be considered because of
the high risk of possible bleeding complications.
3. Transjugular Liver Biopsy (TJLB)

TJLB was initially introduced in dogs as an experimental application by Dotter [29]. Rosch
[7, 8] then reported its clinical application for transjugular cholangiography in 1973 and
1975. TJLB eliminates the need to traverse the peritoneal cavity and puncture the liver cap
sule. Furthermore, this technique is a safer biopsy option for patients with massive ascites,
coagulopathy (prothrombin time greater than 3 seconds over the control value), thrombocy
topenia (less then 60,000/cm3), or those undergoing ancillary procedures such as measure
ment of pressures or opacification of the hepatic vein and inferior vena cava. It can also be

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applied for patients in whom PLB has failed, or those with morbid obesity, a small cirrhotic
liver, suspected vascular tumor or peliosis hepatitis, or medical conditions associated with
bleeding disorders such as hemophilia for whom PLB is contraindicated [11, 30, 31], as any
bleeding is returned to the venous system rather than leaking into the abdomen.
However, there are several particular complications associated with TJLB, including hemor
rhage, subcapsular or neck hematoma and ventricular arrhythmia. The rate of such compli
cations ranges from 0% to 20% [11]. Hardman et al. [4] reported a large subcapsular
hematoma caused by TJLB requiring embolization and prolonged admission. Lebrec et al.
[9] also reported a fatal case of intraperitoneal hemorrhage due to perforation of the liver
capsule caused by excessive of the needle. Therefore, such forward rotation must be avoided
or carefully limited. Furthermore, there have been several direct instances of perforation of
the liver capsule that resulted in aspiration of ascitic fluid, bile from the gallbladder, or renal
tissue in patients with a small cirrhotic liver. In such patients, TJLB should be avoided or
employed only with caution by advancing the needle into the liver parenchyma by only 1
cm instead of the usual 2 cm, or contrast medium should be injected after the biopsy to eval
uate the integrity of liver capsule. The major drawback of TJLB is the size of the biopsy
specimens obtained; they are generally smaller (p <0.001) and more fragmented (p <0.01)
than those obtained by PLB [12]. Pathologically, in terms of the number of portal tracts (p
<0.0001) and the utility of specimens for histological evaluation (p <0.05), the quality of TJLB
samples appears to be significantly lower compared than those of PLB and LLB specimens
[14]. With regard to technical success rate, that of TJLB (82%: 84/102) is significantly lower
(P=0.005) than PLB (100%: 100/100) or LLB (99%: 111/112) [14]. However, Bull et al. [10] re
ported a success rate of 97% (188/197) in 1983, and a recent meta-analysis including more
than 7500 cases revealed a technical success rate of 96.8% [13]. These reports suggest that
there is no significant difference between TJLB and others techniques in terms of success
rate. The most common reason for failure was inability to catheterize the right hepatic vein.
In actual practice, TJLB requires a longer procedure time (40 min) than PLB. A few deaths
after TJLB have been reported, with a mortality rate of 0-0.5% [10, 32, 33]; mortality was due
to hemorrhage from the liver or ventricular arrhythmia.
Therefore, TJLB should be attempted only by a skilled interventional radiologist or physi
cian experienced in catheterization and cannulation of the internal jugular vein due to its
more time-consuming nature, use of intravenous contrast, and the need for a dedicated fluo
roscopy suite. In fact, TJLB can be valuable in cases for which PLB is hazardous, or when
pressure measurement or venography is also required [34]. Despite the smaller biopsy sam
ples obtained, the impact of TJLB on clinical decision-making appears to be comparable to
that of PLB and LLB. In particular, it may help to determine the need for liver transplanta
tion in patients with acute liver failure.
4. Laparoscopic Liver Biopsy (LLB)

There are several approaches for LLB, including PLB under laparoscopic observation, LB
through an additional port under laparoscopic observation, or LB combined with another
25
26
laparoscopic procedure. LLB allows direct observation of the biopsy site and yields with
macroscopic information about the liver surface. This facilitates an adequate sample volume
to be obtained, including wedge resection, without sampling error, and also allows laparo
scopic confirmation of hemostasis. These are the advantages of LLB in comparison with
PLB. If bleeding from the biopsy site persists, compression or coagulation can easily be ap
plied using several types of special forceps.
However, LLB requires general anesthesia and specialized equipment, including insuffla
tion devices and laparoscopic instruments. On the other hand, PLB under laparoscopic ob
servation can be done under local anesthesia using pneumoperitoneum under sedation
using midazolam and disoprivan, or under general anesthesia using an abdominal wall lift
method [15]. For laparoscopy, pneumoperitoneum is created by N2O insufflation via a Ver
ess needle, generally inserted to the left of the umbilicus. A second port is added on the
right side by inserting a trocar. A 16-gauge True-cut needle is inserted and biopsy samples
of the liver can be taken from the left and right lobes under laparoscopic guidance. The bi
opsy sites can be prophylactically coagulated. Beckmann et al. [14] reported that the compli
cations observed after LLB were bleeding and bile leakage, and that the complication rate
(2.7%) was roughly equal to that of PLB (3%) and TJLB (2.9%).
In general, LLB requires a long set-up time for starting the procedure, gas insufflation to cre
ate an adequate operative field, preparation of several laparoscopic instruments, and an op
erating theater. LLB is the most appropriate method for patients who need both a
pathological diagnosis of liver dysfunction or tumor and laparoscopic procedures for intraabdominal diseases.
5. Transgastric Liver Biopsy (TGLB)

For TGLB, Hollerbach et al. [17] have reported an endoscopic ultrasound-guided fine-needle
aspiration biopsy procedure for liver lesions. This method is one of several transgastric ap
proaches and can be an alternative to PLB, particularly for patients with a risk of bleeding or
small lesions in the liver, although targeting may be limited according to tumor location.
Recently, natural orifice translumenal endoscopic surgery (NOTES) has been introduced,
creating no skin scars and involving only minimal invasiveness. NOTES has created a new
access route (via the stomach) to the peritoneal cavity. TGLB using NOTES creates no dam
age to the outside of the body and allows direct observation of the biopsy site inside the
body, unlike PLB or TJLB. In an experimental study, Mintz et al. [35, 36] reported successful
LB using a hybrid technique that included standard laparoscope vision and surgical endos
copy. As outlined in a white paper from the American Society for Gastrointestinal Endos
copy and Society of American Gastrointestinal and Endoscopic Surgeons [37, 38], for clinical
application of NOTES, it is necessary to establish safe access to the peritoneal cavity, com
plete closure of the access route, prevention of infection, correct intra-abdominal orientation,
development of a multitasking platform, methods for management of accidental complica
tions, awareness of unanticipated physiologic events, and training in the technique. In par

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ticular, infection or bacterial contamination in the abdomen due to opening of the digestive
tract is a great concern in NOTES. However, no studies have quantified the bacteriological
load to which the peritoneum is exposed during transgastric procedures [19]. Steele et al.
[20] reported a pilot feasibility study of transgastric peritoneoscopy and liver biopsy during
laparoscopic Roux-en-Y gastric bypass. LB was performed from segment II, III or IVb of the
liver to obtain tissue samples adequate for histologic examination. None of patients exhibit
ed any signs or symptoms of intra-abdominal or trocar wound infection after the procedure.
For TGLB [39], under general anesthesia a forward-viewing, double-channel endoscope is
advanced into the stomach. Puncture of the gastric wall is performed with a 3-mm cuttingwire needle knife. The puncture site is enlarged to 8mm with a balloon dilator and then the
endoscope is advanced into the peritoneal cavity. The peritoneal cavity is then inflated with
air through the endoscope. The liver is easily visualized by retroflexion of the endoscope. LB
is performed using routine biopsy forceps from the edge of the liver (segment III) (Fig. 1),
and hemostasis of the biopsy site is achieved by electrocautery with biopsy forceps (Fig. 2).
The gastric artificial orifice is then closed using endoscopic clips.
Figure 1. Liver biopsy was performed using routine biopsy forceps from the edge of the liver.
Transgastric peritoneoscopy developed by Kalloo et al. [16, 18] showed no association with
serious infection or other complications in the peritoneal cavity during long- term observa
tion. Furthermore, Hazey et al. [40] reported that although contamination of the peritoneal
cavity was observed during laparoscopic Roux-en-Y gastric bypass, no clinically significant
episode, such as abscess formation or infectious complications, occurred. From these find
27
28
ings, although peroral TGLB requires the creation of an artificial injury in a normal organ, it
will likely become a widely used alternative to other LB methods.
Figure 2. Hemostasis was confirmed at the site of liver biopsy.
6. Conclusion
In conclusion, TGLB is technically feasible and has the potential to become an alternative to
routine liver biopsy. The transgastric endoscopic approach has a wide range of diagnostic
and therapeutic applications.
Author details
Nobumi Tagaya*, Nana Makino, Kazuyuki Saito, Takashi Okuyama,
Yoshitake Sugamata and Masatoshi Oya
*Address all correspondence to: tagaya@dokkyomed.ac.jp
Department of Surgery, Dokkyo Medical University Koshigaya Hospital, Koshigaya, Saita
ma, Japan

http://dx.doi.org/10.5772/52914
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outpatient percutaneous liver biopsy. Hepatology 23: 1079-1983.
[2] Sporea I et al. (2008). Why, who and how should perform liver biopsy in chronic liv
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[3] Alotaibi M et al. (2010). The positive color Doppler sign post biopsy: effectiveness of
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s00247-010-1848-7].
[4] Hardman RL et al. (2010). Single-institution results of image-guided nonplugged per
cutaneous versus transjugular liver biopsy. Cardiovasc Intervent Radiol [DOI
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[5] EI-Shabrawi et al. (2012). Outpatient blind percutaneous liver biopsy in infants and
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[6] Szymczak A et al. (2012). Safety and effectiveness of blind percutaneous liver biopsy:
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[7] Rosch J et al. (1973). Transjugular approach to liver biopsy and transhepatic cholan
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[8] Rosch J et al. (1975). Transjugular approach to the liver, biliary system, and portal cir
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[9] Lebrec D et al. (1987). Transvenous (transjugular) liver biopsy. An experience based
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[10] Bull HJM, et al. (1983). Experience with transjugular liver biopsy. Gut 24: 1057-1060.
[11] McAfee JH et al. (1992). Transjugular liver biopsy. Hepatology 15 (4): 726-732.
[12] Meng HC et al. (1994). Transjugular liver biopsy: comparison with percutaneous liv
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[13] Keshava SN, et al. (2008) Transjugular liver biopsy: What to do and what not to do.
Ind J Radiol Imaging 18: 245-248.
[14] Beckmann MG, et al. (2009). Clinical relevance of transjugular liver biopsy in com
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[15] Chiesa OA, et al. (2009). Isobaric (gasless) laparoscopic liver and kidney biopsy in
standing steers. Can J Vet Res 73 (1): 42-48.
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[16] Kalloo AN et al. (2000). Flexible transgastric peritoneoscopy: a novel approach to di

agnostic and therapeutic interventions in the peritoneal cavity [abstract]. Gastroen
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[17] Hollerbach S, et al. (2003). Endoscopic ultrasound-guided fineneedle aspiration bi
opsy of the liver: histological and cytological assessment. Endoscopy 35 (9): 743-749.
[18] Kalloo AN et al. (2004). Flexible transgastric peritoneoscopy: a novel approach to di
agnostic and therapeutic interventions in the peritoneal cavity. Gastrointest Endosc
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[19] Babatin MA et al. (2007). NOTES: Evolving trends in endoscopic surgery. Saudi J
Gastroenterol 13 (4): 207-210.
[20] Steele K et al. (2008). Flexible transgastric peritoneoscopy and liver biopsy: a feasibili
ty study in human beings (with video). Gastrointest Endosc 68 (1): 61-66.
[21] Vijayaraghavan GR et al. (2011). Imaging-guided parenchymal liver biopsy: How we
do it. J Clin Imaging Sci [DOI: 10.4103/2156-7514.82082].
[22] Piccinino F et al. (1986). Complications following percutaneous liver biopsy. A multi
centre retrospective study on 68,276 biopsies. J Hepatol 2: 165-173.
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[24] McGill DB et al. (1990). A 21-year experience with major hemorrhage after percutane
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nationwide survey. For the Group of Epidemiology of the French Association for the
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[26] Pasha T et al. (1998). Cost-effectiveness of ultrasound-guided liver biopsy. Hepatolo
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[27] Younossi ZM et al. (1998). Ultrasound-guided liver biopsy for parenchymal liver dis
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[28] Farrell RJ et al. (1999). Guided versus blind liver biopsy for chronic hepatitis C; clini
cal benefits and costs. J Hepatol 30: 580-587.
[29] Dotter CT. (1964) Catheter biopsy: experimental technique for transvenous liver bi
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[30] Bravo AA et al. (2001). Liver biopsy. N Engl J Med 344: 495-500.
[31] Rockey D et al. (2009). American Association for the Study of Liver Diseases. Liver
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[33] Kalambokis G et al. (2007). Transjugular liver biopsy-indications, adequacy, quality

of specimens, and complications: A systematic review. J Hepatol 47: 284-294.
[34] Gilmore IT, et al. (1977). Transjugular liver biopsy. Br Med J 9: 100-101.
[35] Mintz Y et al. (2007). NOTES: The hybrid technique. J Laparoendosc Adv Surg Tech
17: 402-406.
[36] Mintz Y et al. (2008). NOTES: A review of the technical problems encountered and
their solutions. J Laparoendosc Adv Surg Tech18: 583-587.
[37] ASGE & SAGES. (2006). ASGE/SAGES working group on natural orifice translume
nal endoscopic surgery: White paper October 2005. Gastrointest Endosc 63: 199-203.
[38] Rattner D & Kalloo A: ASGE/SAGES Working Group. (2006). ASGE/SAGES working
group on natural orifice translumenal endoscopic surgery. October 2005. Surg En
dosc 20: 329-33.
[39] Tagaya N, et al. (2010). Transgastric liver biopsy using the NOTES technique: an ani
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171-178.
[40] Hazey JW et al. (2008). Natural-orifice transgastric endoscopic peritoneoscopy in hu
mans: Initial clinical trial. Surg Endosc 22: 16-20.
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Chapter 3
Rethinking the Role of

Liver Biopsy in the Era of Personalized Medicine
http://dx.doi.org/10.5772/53120
1. Introduction
Indications and methods of liver biopsy have changed over the past few years [1]. However,
an histological diagnosis may be needed for optimal management of a patient [2, 3].
Although modern biochemical, immunological, and radiographic techniques have facilitat
ed the diagnosis and management of liver diseases they have not made liver biopsy obso
lete. Clinicians rely on information derived from the liver biopsy to inform patients and to
make their therapeutic options [4].
There are, however, many controversies surrounding liver biopsy resulting potential limita
tions, such as sampling errors and interobserver variations [5], which can lead to misclassifi
cation therefore, P. Bedossa et al. consider that when it comes to liver biopsy the term best
standard is more appropriate than gold standard [6].
It is essential, when analysing the indications, contraindications, complications and other as
pects of the liver biopsy, to consider present hepatology and personalized medicine.
Practiced since the late 19th century, liver biopsy remains the criterion standard in the
evaluation of the etiology and extent of disease of the liver. Paul Ehrlich performed a per
cutaneous liver biopsy in Germany in 1883. [7]. Since then, this method has been im
proved with the introduction of different needle types for cutting and aspiration. But,
until the 1950s, when Menghini developed an aspiration technique which led to a wider
use of the procedure and broadened its applications, it was not common. While in the ear
ly 1960 and 1970s the liver biopsy was used for making a diagnosis in cases of suspected
medical liver disease, today it is more often performed to assess the prognosis or evaluate
therapeutic strategies [1].
34
With regards to the technique used to carry out the liver biopsy there has also been a major
change, it used to be performed blindly by clinicians, specialists in gastroenterology or hep
atology at the patients bed whereas at present, percutaneous biopsies are performed pri
marily by radiologists.
Currently, a liver biopsy can be obtained either transvenously or transcutaneously, or by
combining imaging modalities such as ultrasound, computed tomography, and laparosco
py. The choice of one technique over another is based on availability, personal preference,
and the clinical situation.
Liver biopsy techniques: Percutaneous, transjugular or laparoscopic
Percutaneous liver biopsy can be transthoracic, with an intercostal liver access or subcos
tal, when the patient has an enlarged liver extending below the costal margin. Clinicians
have now discarded blind liver biopsies in favour of ultrasound-guided biopsies.
Transjugular or transvenous liver biopsy was first described in 1964. It is a technique used
in order to avoid percutaneous liver biopsy in patients who are at a higher risk of bleed
ing. However, it has its limitations and is considered an inferior biopsy due to the frag
mentation of the obtained specimen, which may reduce the accuracy of the diagnosis. It is
performed in a vascular catheterisation laboratory by a radiologist with special training in
interventional radiology. Videofluoroscopy equipment and cardiac monitoring are man
datory due to the risk of cardiac arrhythmia as the catheter passes through the right at
rium. With this method, hepatic venography, wedged hepatic venous pressure, caval
pressure and atrial pressure measurements can also be obtained during the procedure.
The most frequent indications for the transjugular route are: severe coagulopathy, ascites,
obesity, suspected vascular tumour or peliosis hepatis.
Laparoscopic liver biopsy. This technique is well established and its use varies between
centers. It is indicated in centers where access to transvenous liver biopsy is not available,
and in patients with focal liver lesions and coagulopathy for whom obtaining histology is
essential for their management.
The decision to use a particular technique is based on the risk profile of the patient. If he or
she has advanced liver failure with coagulopathy and ascites, liver biopsy is unnecessary,
but the diagnosis of the underlying disease is crucial in specific circumstances in order to
determine a therapy, for example in cases of liver transplant. Before a liver biopsy it is nec
essary to carry out an ultrasound to quantify vascular permeability and because it may rule
out anatomical abnormalities and can identify mass lesions that are clinically silent. When
cirrhosis is suspected on clinical grounds, or by non-invasive methods liver biopsy is usual
ly avoided.
2. Contraindications to percutaneous liver biopsy

Absolute contraindications: the main contraindication to percutaneous liver biopsy is
significant coagulopathy, others are: uncooperative patient, history of unexplained bleed
Rethinking the Role of Liver Biopsy in the Era of Personalized Medicine

http://dx.doi.org/10.5772/53120
ing, prothrombin time 35 seconds more than control, platelet count less than 50,000/
mm3, the use of a non-steroidal anti-inflammatory drugs, (unless discontinued 7 to 10
days previously), blood for transfusion unavailable, suspected hemangioma, another vas
cular tumor or echinococcal cysts in the liver, and the inability to identify an appropriate
site for biopsy.
Relative contraindications: Morbid obesity, ascites, hemophilia, infection in the right pleu
ral cavity or below the right hemidiaphragm.
Accepted indications: Given the new developments that have proved the efficacy of liver
biopsy, its role in the management of patients with chronic liver diseases has much evolved
in recent years and will continue to evolve as new non invasive technologies are developed.
Diagnosis
1. Many parenchimal liver diseases
2. Abnormal liver tests
3. Fever of unknown origin
4. Focal or diffuse abnormalities on imaging studies
Prognosis-Staging of known parenchimal disease
Management Developing treatment plans based on histologic analysis
Contraindications for percutaneous liver biopsy
Absolute: uncooperative patient, severe coagulopathy, infection of the hepatic bed, extrahepatic biliary obstruction.
Relative: ascites, morbid obesity, possible vascular lesions, amyloidosis, hydatid disease.
Table 1. Indications and contraindications for liver biopsy
Its importance in diagnosis, staging and prognosis largely depends on the indication and the
clinical question relying on an answer from the histological result.
2.1. Is liver biopsy always necessary?
The utility of routine liver biopsy has been the subject of debate in recent years. Due to liver
biopsy being associated with a small but definite risk, a biopsy should only be performed
when the findings contribute to a better management of the patient. It is argued that for the
purposes of management, liver biopsy is neither needed in cases with advanced fibrosis nor
those diagnosed with cirrhosis by other methods, nor in patients with mild disease, for
whom a therapeutic decision is not urgent. Until recently, liver biopsy played a key role in
the evaluation of chronic liver disease, but now in the presence of better diagnostic tests on
disease etiologies and treatments its role has to be re-evaluated. Recognition and confirma
tion of the pattern of injury (chronic hepatitic, chronic cholestatic, steatohepatitic, etc.) is the
pathologist's priority when evaluating the liver biopsy.
35
36
Moreover, liver biopsy provides information on the severity and distribution of lesions (co
dified in the staging and grading of chronic liver disease), the presence of confounding pat
terns of injury (such as steatohepatitis coexisting with chronic viral hepatitis), and the
presence of additional findings such as steatosis or iron accumulation that may have prog
nostic or therapeutic relevance.
2.2. Who should be biopsied?
As a rule patients with standard clinical and radiological features are not biopsied. Howev
er, in the presence of non concordant or atypical results, a biopsy may be recommended.
The decision whether to perform a liver biopsy in some patients is clear, however in cases
with a suspected concomitant diagnosis or when results from other methods are non conclu
sive confirmation is needed [8,9].
Type of Injury
Causes
Fatty change
Ethanol, fatty liver disease, obesity, diabetes, drugs
Councilman bodies
Viral hepatitis, drugs, toxins, ischemia (acidophilic bodies)
Mallory bodies * (hyaline)
Ethanol, obesity, diabetes, drugs, Wilson disease, biliary
(see pages: 13, 15, 17 and 18)
tract disease, hepatocellular carcinoma
Hydropic change (ballooning degeneration)
Viral hepatitis, drugs, cholestasis, fatty liver disease
Cholestasis
Duct obstruction or injury, drugs, viral hepatitis
Interlobular duct injury
Primary biliary cirrhosis, primary sclerosing cholangitis,

hepatitis C
Piecemeal necrosis
Viral hepatitis, primary biliary cirrhosis, drugs, Wilson

disease
Increased iron stores
Hemochromatosis, transfusions, hemolysis
Granulomas**
Sarcoid, infections (tuberculosis, fungi), drugs
(see pages: 19, 20 and 21)

Table 2. Patterns of liver cell injury found in liver biopsies and clinical differential diagnosis
3. The generally accepted indications of liver biopsy are the following

Diagnosis for a better scoring of grading and staging of chronic viral hepatitis C or hepati
tis B, alcoholic liver disease, non-alcoholic steato-hepatitis, or autoimmune hepatitis.
In patients with raised ferritine for the diagnosis of hemochromatosis
If there are suspected disorders of cupper metabolism for the diagnosis of Wilsons dis
ease, with quantitative estimation of copper in liver tissue.
Evaluation of possible autoimmune hepatitis

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In cholestatic liver diseases: primary biliary cirrhosis, primary sclerosing cholangitis and
overlap syndromes.
Evaluation of abnormal results of biochemical tests of the liver in association with a sero
logic workup that is negative or inconclusive
Evaluation of the efficacy or the adverse effects of treatment regimens (e.g.,methotrexate
therapy for psoriasis).
Alcohol related disease. Non-alcoholic fatty liver disease (NAFLD) or Non-alcoholic stea
tohepatitis (NASH).
Diagnosis of a liver mass, in selected cases, when image tests are inconclusive.
Evaluation of fever of unknown origin, with an eventual culture of liver tissue.
Evaluation of the status of the liver post transplantation or of the donor liver pre trans
plantation.
4. Methods: How to handle a liver biopsy

Liver samples should be fixed in 10% neutral buffered formalin because this enables all rou
tine histochemical and immunohistochemical staining to be carried out. A small portion of
the sample could be snap-frozen for adjunctive molecular studies for diagnostic or research
purposes, particularly when multiple etiologies are clinically suspected.
As for stains, a good collagen stain to assess fibrosis is mandatory. Perls stain for iron is rec
ommended and the Periodic Acid-Schiff (PAS) stain with and without diastase digestion is
useful for assessing hepatocyte cytoplasm glucogen content.
Special stains for special circumstances are ordered if indicated by the clinical situation. For
instance, the Ziehl-Nielsen is ordered for mycobacteria, and Grocotts silver methanamine
stain is used when granulomas are observed or when fungi infection is suspected. The Con
go Red stain is requested when amyloid is suspected to be present Rhodamine stain, Victo
ria blue or orcein stain is used to detect copper deposition when there is clinical suspicion of
Wilsons disease. Immunohistochemistry is used to confirm the presence of Hepatitis B sur
face antigen and Hepatitis B core antigen [10].
Cultures could be indicated in selected cases such as Mycobacterias [11].
5. Histologic diagnosis and clinical correlation

The pathological report that used to be too descriptive now has to include etiology, aspects
related to prognosis and possible therapy [12,13].
In order to promote the clinico-pathological diagnostic correlation with the intention of im
proving communication and clarifying individual cases, regular meetings between clinicians
37
38
and pathologists are necessary. It is not only important to hold formal conferences but also to
increase daily exchanges. To facilitate the communication between pathologists, radiologists,
surgeons and clinicians it is desirable, when feasible, for the same teams to work together.
6. Writing the histology report

In order to produce a clinically relevant liver histology report pathologists should follow the
internationally accepted guidelines.
1.
The adequacy of the biopsy should be assessed by measuring the length of the speci
men and counting the number of portal tracts. The data should be written up in the fi
nal report to make clinicians aware of any potential sampling error in the grading and
staging. To reduce sampling error the amount of tissue required is usually 1 to 4 cm
long and needs to include at least four portal tracts.
2.
The type and severity of necroinflammation and fibrosis should be described in words.
By only using numbers to report the presence or not of bridging necrosis for example,
some clinically useful information might be omitted. A validated scoring system should
be used for grade of activity and stage of fibrosis.
3.
As well as being described, the existence of adjunt data should be scored subjectively,
such as steatosis graded on a scale of 0-3 and siderosis graded on a scale of 0-4.
4.
Other diagnostic criteria may be useful in differential diagnoses:

Differentiating viral hepatitis from other chronic disorders, such as cell dysplasia and
thus separately reporting the presence of cell changes.
Using immunostaining when appropriate, for example Hepatitis B Virus antigens
[14].
Searching for any concomitant diseases.
5.
Chronic viral hepatitis, primary biliary cirrhosis and autoimmune hepatitis have typical
histological lesions and it is advisable to consider the characteristics of: portal tract in
flammation, interface hepatitis, lobular necrosis and bile duct damage, separately [15].
6.
Concomitant histological features in liver specimens of hepatitis C cases: auto-immuni

ty, co-infections, steatosis, hemosiderosis, malignancy related changes, hepatitis due to
drugs and/or vascular problems. Furthermore, biopsy frequently detects associated le
sions such as steatosis or steatohepatitis providing information related to management
and prognosis of patients with chronic hepatitis C.
7.
Finally the conclusions should be written in order to make the histological diagnosis,
stating whether the pathological findings are consistent with chronic hepatitis or not,
whether a specific viral etiology may be suspected or whether there are changes related
to concomitant diseases, specifying which.

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Chronic viral hepatitis

Portal tract
Primary Biliary Cirrhosis (PBC)
Mononuclear cells
Mononuclear cells; eosinophils
Common
Common;ductular reaction
inflammation
Interface hepatitis
Autoimmune Hepatitis
Mononuclear cells;
plasma cells
Present
(i.e. biliary piecemeal necrosis)

Lobular necrosis
Variable degree usually focal
Variable; usually mild and focal
Severe, may be confluent
Bile duct damage
Common in hepatitis C
Duct destruction present
May be present
(usually mild)
Table 3. Specific features in liver biopsy differential diagnosis and pathological findings
7. Use of liver biopsy in clinical practice

Here we will discuss some of the most prominent findings of liver biopsy in the following
clinical settings:
a.
Viral Hepatitis : Hepatitis B, Hepatitis C.
b.
Metabolic diseases: Hemochromatosis, Porphyria, Wilson disease, Alpha 1- anti-trypsin

disease.
c.
Autoimmune and cholestatic diseases: Auto-Immune Hepatitis, Primary Biliary Cirrho

sis (PBC), Primary Sclerosing Cholangitis (PSC), Overlap Syndrome.
d.
Alcoholic liver disease
e.
Non-alcoholic Fatty Liver Diseases and Non-Alcoholic Steato-Hepatitis (NAFLD and

NASH). Liver steatosis.
f.
Drug-Induced Liver Injury (DILI).
g.
Infections and pyrexia of unknown origin.
h.
Cirrhosis. Fibrosis progression.
i.
Hepatocellular Carcinoma (HCC) and other benign or malignant focal lesions. The role
of the fine neddle aspiration biopsy (FNAB) and other imaging diagnostic tools.
j.
New evolving fields for liver biopsy: Liver transplantation and living donors. Bone
marrow transplantation. Morbid obesity
7.1. Chronic viral hepatitis C and B

In the past the majority of liver biopsies were performed in chronic hepatitis C patients.
However, recently this has changed, due to a better understanding of the etiology, patho
genesis, the natural history of the disease and available therapies.
39
40
As the ability to treat hepatitis C effectively improves, the value of information gained from a
liver biopsy decreases. The most effective therapy currently available, a combination of pegy
lated interferon and ribavirin, can induce sustained viral clearance, implying a definitive
cure and improved long term prognosis. This occurs, after anti-viral treatment in up to 80% of
patients infected with genotypes 2 and 3. In patients with genotype 1 receiving recently ap
proved telaprevir and boceprevir, triple therapy constituents, an average of 70-75% can ach
ieve sustained viral clearance. Due to the high percentage of positive response in persons with
genotypes 2 and 3, the need for a liver biopsy in such cases has been questioned.
The terminology used to assess the appearance of liver biopsies with chronic viral hepatitis
has also evolved.
The first classification of chronic hepatitis based on histological criteria was published in
1968. At that time, only three diseases causing chronic hepatitis could be diagnosed, hepati
tis B, non A-non B (hepatitis C, since 1989) and autoimmune hepatitis. This classification
which also had prognostic implications only had two categories, namely "chronic persistent
hepatitis" and "chronic active hepatitis". Three years later, the term chronic lobular hepatitis
was added to represent findings similar to those observed in acute hepatitis.
During the 1990s, there were great changes in the understanding of chronic viral hepatitis
by pathologists and hepatologists. The new concepts recognized that the traditional catego
rization of pathologic changes (chronic persistent hepatitis, chronic lobular hepatitis, and
chronic active hepatitis) was inadequate for assessing histological changes during clinical
trials. Pathologic processes were separated rather than considered as part of a continuum of
pathologic changes that occur in chronic hepatitis C. Pathologists introduced the idea of
staging for fibrosis and grading for the inflammatory component to the pathological evalua
tion of chronic hepatitis C.
7.1.1. Grading and staging of chronic hepatitis C Scoring Systems (Table 4) [16-20]
Grading is the assessment of the activity of a disease, which may increase and decrease as a
disease flares and subsides, or may remain static throughout the disease.
Grade and stage evaluation is a standard part of the pathologic assessment of liver biopsies in
chronic hepatitis. Pathological staging has focused on the assessment of fibrosis as the best
surrogate marker of the disease process. Staging divides the fibrotic continuum into discrete
categories and all of the existing staging systems have cirrhosis as their highest stage. Several
systems exist for grading and staging of chronic hepatitis and all have been used effectively to
assess changes in pathology following therapeutic intervention. These systems include the
methods of Scheuer, Desmet, Batts and Ludwig,and the METAVIR system used to score indi
vidual features of inflammation and fibrosis semi-quantitatively in clinical studies [14-20].
Steatosis and Steatohepatitis in chronic hepatitis C Steatosis in hepatitis C is mainly macro
vesicular and a common finding in genotype 3 [21] it is also related to a high body mass in
dex and older age. More recently, steatosis has been recognized as a feature worthy of
study, from an etiologic standpoint and especially in terms of its clinical significance. Esti
mation of the degree of steatosis has been hampered by the lack of standard definitions and

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breakpoints between grades. Although the intrinsic mechanism and involved factors for ac
celerated fibrosis are unclear, steatosis has been associated with increased inflammation,
hepatocellular apoptosis and the presence of perisinusoidal fibrosis [22].
Utility of biopsy in hepatitis C. Nowadays, the majority of Hepatitis C patients can be man
aged without having to undergo a liver biopsy since liver biopsy rarely identifies unsuspect
ed etiology and hepatitis C diagnosis relies on blood antibody and HCV RNA
determinations. However, a biopsy allows to identify patients most in need of therapy or to
find clinically unsuspected cirrhosis, which when found it is necessary to screen for varices
and hepatocellular carcinoma.
Moreover clinical and laboratory surrogates for biopsy may be useful in identifying cirrho
sis and biopsy is not necessary if clinical, image and analytical data concur. Post-treatment
biopsy is not needed, nevertheless a new liver biopsy, could be performed if new treatments
or clinical trials arrive in order to stratify patients by prognosis.
Fibrosis stage Knodell et al.
Scheuer, 1991
METAVIR, 1994
Batts and
1981 [16]
[17]
[18]
Ludwig, 1995
No Fibrosis
No Fibrosis
No Fibrosis
Fibrous portal
Enlarged fibrotic
expansion
portal tracts
Ishak et al. 1995 [20]
[19]
No Fibrosis
No Fibrosis
Portal fibrosis
Fibrous expansion of
some portal areas, with or
without short fibrous
septa
Periportal or
Enlargement of
Periportal
portal-portal
portal tracts
fibrosis
most portal areas, with or
septa but intact
without short septa
architecture
3
Bridging
Fibrosis with
Fibrosis (portal-
architectural
Septal fibrosis Fibrous expansion of

most portal areas with
portal or portal- distortion but no

4
central linkage
obvious cirrhosis
Cirrhosis
Probable or
definite cirrhosis
occasional portal to
portal bridging
Cirrhosis
portal areas with marked
bridging (portal to portal
as well as portal to
central)
Marked bridging with

occasional nodules
(incomplete cirrhosis)
Cirrhosis probable or
definite
Table 4. Comparison of commonly used scoring systems for fibrosis staging in chronic Hepatitis C
41
42
7.1.2. Natural history

The degree of inflammation, fibrosis stage, and steatosis seen on liver biopsy are key histo
logical predictors of progression to cirrhosis. (Table 5) (see page 28).
Based on retrospective data, it has been shown that most patients with moderate inflamma
tion on initial liver biopsy developed cirrhosis after 20 years, and nearly all patients with se
vere inflammation or bridging fibrosis developed cirrhosis in 10 years. Patients with mild
inflammation and/or minimal fibrosis have a low risk of progression to cirrhosis. Hepatic
steatosis is also an emerging risk factor for fibrosis progression in hepatitis C [22]. Clinical
information may help to refine prognosis, but cannot substitute the valuable information ob
tained from a liver biopsy. Poynards group showed three clinical factors which are inde
pendently associated with faster progression of fibrosis: male, aged over 40 at the time of
infection, and having a daily alcohol consumption of 50 grams or more [23]. Other factors
predicting progression to cirrhosis include immunosuppression and co-infection with hepa
titis B or HIV [24].
Utility of biopsy in hepatitis B Liver biopsy is not mandatory but may show moderate or
severe inflammation which is why before starting antivirals, usually for a long period, our
protocol is to perform a liver biopsy and to individualize the therapeutic decision [25]. It has
been proved that long-term therapy may improve histology but the role of serial liver biop
sies has yet to be established outside of clinical trials. Fibroscan has yet to be validated for
patients with chronic hepatitis B but research on this is ongoing [26].
It is important to identify cirrhosis to indicate anti-hepatitis B therapy and hepatocellular
carcinoma screening is also recommended for all hepatitis B surface antigen-positive
(HBsAg+) patients, cirrhotic or not. New guidelines on anti-HBV treatment say that it is ad
visable to treat patients with elevated DNA-HBV and minimally elevated or fluctuating ala
nine aminotransferase (ALT), [27, 28].
Features typical of chronic viral hepatitis inflammation, like fibrosis, is considered to be one
of the key characteristics of chronic viral hepatitis. It is a chronic necroinflammatory process
in which hepatocytes are preferentially injured compared with bile ducts. The grade of in
flammation is a stratification of the overall necroinflammatory changes into mild, moderate,
and marked categories. Unlike the fibrosis systems, which are based on distinctive architec
tural changes that can be highlighted with special stains, assessment of inflammation is
more subjective and hence shows more interobserver variations. Usually varying degrees of
portal and periportal inflammation(with lymphocytes, plasma cells, and macrophages), lo
bulillar hepatitis, and fibrosis are to be individually considered and scored.
Interface hepatitis occurs when the inflammatory infiltrate crosses the limiting plate; it is
usually associated with local hepatocyte damage, piecemeal necrosis, and inflammation.
Lobular inflammation is accompanied by some hepatocellular necrosis (acidophilic or
Councilman bodies). Chronic hepatitis leads to progressive fibrosis and, without treat
ment, to cirrhosis. The fibrosis begins in portal areas, extends to periportal areas, bridg
ing also other portal tracts and central veins. Histopathological findings in the liver

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biopsy that help to predict etiology chronic hepatitis B may show some of the changes
described previously, as well as a ground-glass change to the cell cytoplasm. This
change reflects accumulation of hepatitis B surface antigen within the endoplasmic retic
ulum of the hepatocytes [29].
Chronic hepatitis C may be associated with prominent lymphoid aggregates within portal
tracts, sometimes including germinal centers and, occasionally, bile duct damage, although
not to the degree seen in line primary biliary disorders. In addition, biopsies may show fo
cal, nonzonal macrovesicular steatosis [30].
Patterns of liver cell injury found in liver biopsy and differential diagnosis. Chronic viral
hepatitis have no unique histopathologic features, it is therefore necessary to consider vari
ous causes. In addition to viral infection, chronic hepatitis may be autoimmune or drug re
lated. Histological features of chronic cholestatic disease, including PBC, primary sclerosing
cholangitis (PSC), autoimmune cholangitis, as well as metabolic diseases including Wilson
disease and 1-antitrypsin deficiency, may overlap with some of the findings with so
called chronic hepatitis.
7.2. Metabolic liver diseases
Many rare diseases originate in the liver, either affecting the liver directly or causing extrahepatic disease [31]. For example, liver histology is usually normal in primary hyperoxaluria
while the kidneys and other organs may be irreparably damaged; however, cure is only pos
sible with a liver transplant. In other inherited disorders, the liver disease may remain
asymptomatic until precipitous acute liver failure develops; the classic example is Wilson
disease. Here we present the diseases most frequently observed in adult patients.
7.2.1. Hematochromatosis: The role of liver biopsy in the diagnosis of hepatic iron overload in the era
of genetic testing [32]
Hemochromatosis is an autosomal recessive disorder that leads to massive deposits of iron
in many organs, including liver, pancreas, heart, joints, and skin. The gene responsible for
hereditary hemochromatosis, HFE, is located on chromosome 6. The two most common mu
tations are C282Y (present in up to 80% of cases) and H63D. The defining characteristic of
this disease is the failure to prevent unneeded iron from entering the circulatory pool as a
result of genetic changes compromising the synthesis or activity of hepcidin, the iron hor
mone.Hemochromatosis results from the interaction between genetic and acquired factors.
Depending on the underlying mutation, the coinheritance of modifier genes, the presence of
nongenetic hepcidin inhibitors, and other host-related factors, clinical manifestation may
vary from simple biochemical abnormalities to severe multiorgan disease [33]. The indica
tion of a liver biopsy in the era of genetic testing is being questioned. But, in our opinion,
liver biopsy continues to play an important role in the diagnosis, prognosis and manage
ment of patients with elevated serum ferritin and abnormal liver function test results in gen
eral hepatology practice. Genetic tests for HFE mutations (C282Y, H63D) and liver biopsies
are complementary in the workup of these patients.
43
44
Liver biopsy allows a quantitative iron concentration study and the identification of the
grade of hepatic iron overload, localization pattern and associated liver pathology for diag
nosis and management of patients [34].
Liver biopsies may be relatively normal or show bridging fibrosis or even micronodular cir
rhosis. Untreated, hemochromatosis leads to the development of micronodular cirrhosis.
Prior to the availability of genetic testing, the diagnosis of hemochromatosis was always de
termined with liver biopsy and quantitation of tissue iron. With the availability of genetic
testing for the C282Y and/or H63D mutations, liver biopsy is more often reserved for evalu
ation of clinical status or complications (i.e. degree of fibrosis, development of hepatocellu
lar carcinoma) rather than for primary diagnosis [35]. A biopsy can also help determine if
other disease processes are present, such as hepatitis C or fatty liver disease [36].
We suggest that patients with suspected hemochromatosis undergo genetic testing for the
C282Y and H63D mutations, especially if they have a family history of hemochromatosis,in
order to establish the genotype of the patient and permit genetic counseling. A liver biopsy
may not be necessary in young C282Y homozygotes or in heterozygotes without evidence of
liver disease.
Disorders that have to be considered in the clinical differential diagnosis of hemochromatosis
The list of disorders associated with increased hepatic iron is long. The majority of patients
with hepatic iron accumulation from any cause do not have hepatic iron concentration (HIC)
that is above the upper limit of normal (approximately 1100 mg/g dry liver weight). Fur
thermore the pattern of distribution of the iron in the liver may be of some help in establish
ing the diagnosis [37]:
predominantly hepatocellular distribution of iron leads to a diagnosis of genetic hemo
chromatosis, alcoholic liver disease and/or porphyria cutanea tarda.
predominant presence of iron in Kupffer cells, may be the result of multiple transfusions
and/or hemolytic anemias.
a mixed distribution of iron may be a sign of megaloblastic anemia or anemia secondary
to chronic infection.
7.2.2. Porphyria Cutanea Tarda (PCT)
It is the most common form of porphyria across the world. PCT is usually an acquired liver
disease caused by exogenous factors, such as excess alcohol intake, iron overload, chronic
hepatitis C and oestrogen therapy.
The pathogenesis of PCT is varied; it may be hereditary or acquired, leading to hepatic iron
loading and to an increase of oxidative stress. Iron loading is usually only mild or moderate
in degree. However, in patients with excessive alcohol intake and/or chronic hepatitis C in
fection, hepcidin production by hepatocytes decreases. This decrease is responsible for in
creased iron absorption from the gut. The important role that PCT often plays in the
hepatitis C virus setting has recently been emphasized [38].

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7.2.3. The role of liver biopsy in determining the diagnosis of Wilson disease
Wilson disease is an autosomal recessive disorder of copper metabolism, characterized by
excessive accumulation of copper in the liver and other organs. Genetic evaluation is diffi
cult because most patients are compound heterozygotes. For patients with Wilson disease
the norm is to perform a liver biopsy with a quantitative copper testing of the liver; levels
are typically greater than 250 mg/g dry weight liver (normal level, 38 mg/g) [39].When the
diagnosis of Wilson disease is considered prior to liver biopsy other tests are undertaken. Serum ceruloplasmin (less than 20 mg/dL in patients with Wilson disease; normal levels, 23
to 50 mg/dL). - 24-hour urinary copper (greater than 100 mg/dL; normal, less than 30 mg/
dL). -Kayser-Flescher ring has to be studied by ophthalmologic testing. The liver biopsy in
this disease can present differently, depending on the patients age. In children and young
adolescents, the most common finding may be fatty change. In older adolescents and young
adults, a liver biopsy may show chronic hepatitis with piecemeal necrosis. Adults tend to
show cirrhosis, and Mallory bodies*. In adolescents or adults, confluent necrosis may lead to
a severe hepatic failure that may require an urgent liver transplant [40].
7.2.4. Alfa1 -antitrypsin (A1-AT) deficiency on liver biopsy
A1-AT is the major circulating inhibitor of serine proteases (Pi). Its primary target is the po
tent elastase found in polymorphonuclear cells (PMNs). It is a glycoprotein synthesized in
the liver. Many of the Pi variants are associated with fairly normal serum concentrations and
function and thus are of little clinical significance. However, a few, result in low circulating
levels of 1-AT (i.e., PiZZ) and are of pathologic significance. Liver biopsies from affected
patients demonstrate classic PAS-positive, diastase-resistant globules within periportal hep
atocytes. Portal fibrosis and chronic hepatitis may also be present. Liver cell dysplasia may
be seen, and patients older than 50, especially men, are at risk of developing hepatocellular
carcinoma. The presence of PAS-positive, diastase-resistant globules is not always diagnos
tic for A1-AT deficiency because various inflammatory conditions may be associated with
overproduction of the enzyme, as is the case in cardiac congestion or hypoxia. For this rea
son clinical correlation is required [41].
7.3. Autoimmune Hepatitis (AIH)
Autoimmune hepatitis (AIH) is an inflammatory condition of the liver that can affect pa
tients of all ages, sexes, and races [42].
Timely diagnosis and immunosuppressive therapy may control disease activity in almost all
affected patients and various case series have reported near normal or normal life expectan
cy in patients diagnosed and treated adequately [43]. Untreated AIH, however, has 5-year
mortality above 50%.
It was first described as a form of chronic hepatitis in young women, showing jaundice, ele
vated gammaglobulins and amenorrhea, which eventually leads to cirrhosis. There is not a
single test to diagnose AIH but a set of diagnostic criteria has been suggested in order to
classify patients as having probable or definite AIH depending on a score.
45
46
Clinical appearance ranges from an absence of symptoms to a severe fulminant presenta

tion. It is usually clinically associated with other autoimmune diseases such as rheumatoid
arthritis, ulcerative colitis, autoimmune thyroiditis, or diabetes mellitus. A family history
has been reported [44].
A liver biopsy should be obtained at first diagnosis before therapy for grading, staging and
confirmation of the diagnosis. Histological appearance is not characteristic, although typical
features such as periportal hepatitis with lymphocytic infiltrates, plasma cells and piecemeal
necrosis, with more advanced disease bridging necrosis, are frequent. Variable degrees of
portal fibrosis are present [45]. In non treated patients or in non-responsive to corticosteroid
therapy cirrhosis eventually occurs.
Differential diagnosis which has been revisited recently by the International Autoimmune
Hepatitis Group comprises: chronic hepatitis not caused by other etiologies (viral, drug-in
duced), acute hepatitis alone or acute hepatitis superimposed on underlying chronic liver
disease and autoimmune diseases with associated duct damage and duct loss [47].
Early diagnosis may be difficult because the clinical picture is heterogeneous and the liver
histology sometimes shows atypical features.
A simple and accurate diagnostic scoring system for AIH has been established but not
totally validated yet. In 1993, the International Autoimmune Hepatitis Group (IAIHG)
proposed specific diagnostic criteria, which were revised in 1999. These criteria were
made by expert consensus and introduced to allow comparison of studies from different
centers [46]. Some of the items were of questionable value which is why in 2008 the
IAIHG published a new simplified scoring system for wider applicability in routine clini
cal practice, based on the data of patients with well-established diagnoses and validated
in another group of patients [47].The new score includes autoantibodies, immunoglobu
lin G, histology, and exclusion of viral hepatitis, allowing a reliable diagnosis of AIH ap
plying simple scores.
7.3.1. Primary Biliary Cirrhosis (PBC)
PBC is a chronic progressive cholestatic liver disease that occurs in middle-aged patients,
usually women, and is often associated with other autoimmune diseases. Patients may
present with jaundice and pruritus in advanced cases. Laboratory testing reveals serum an
timitochondrial antibody (AMA) as well as increased alkaline phosphatase, bilirubin, and
-glutamyl transpeptidase [48]. The histological staging of PBC considers the degree of bile
duct damage and fibrosis [49].
- Stage 1 early disease is characterized by damage to septal and larger interlobular bile
ducts, reflected by biliary epithelial damage with infiltration of the duct by lymphocytes,
plasma cells, eosinophils, and rare polymorphs. The inflammatory infiltrate confined within
the portal tract, may include granulomas and lymphoid follicles (florid duct lesion).
- Stage 2 the inflammatory process extends beyond the portal tract, and changes of interface
hepatitis (piecemeal necrosis) may be seen. Bile ducts begin to disappear and proliferation of

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bile ductules (cholangioles) may also be present along the edges of the portal tracts. These
changes are associated with features of chronic cholestasis, including feathery degeneration
within the cytoplasm of hepatocytes, accumulation of bile pigment, periportal accumulation
of copper (not generalized as in Wilson disease), and, occasionally, Mallory bodies*.
- Stage 3 is associated with increasing fibrosis and bridging between portal areas, with de
creased amounts of inflammation.
- Stage 4 represents biliary cirrhosis, usually micronodular. In the past the diagnosis was
done in very advanced disease, biliary cirrhosis, hence its name.
7.3.2. Primary Sclerosing Cholangitis (PSC)
Primary sclerosing cholangitis (PSC) is a disease with a variable clinical course, with obliter
ation of the biliary tree that leads to biliary cirrhosis and its complications such as portal hy
pertension and liver failure. The term primary is used to distinguish this condition from
the bile duct strictures that are secondary to bile duct injury, cholelithiasis or ischaemia [50].
Patients may present with increased alkaline phosphatase and positive perinuclear anti
neutrophil cytoplasmic antibodies (pANCAs). In this disease, liver biopsy does not have
a crucial role in the diagnosis. Ultrasound is used for the initial investigation and may
show bile duct dilatation and liver and splenic changes; however, it is unspecific for
PSC. [51,52]. The classic lesion of PSC in the histological study is onionskin or concentric
periductular fibrosis, with damage to the ductal epithelium, but it is rarely seen on per
cutaneous biopsy. The most common findings on a biopsy in early-stage disease are non
specific [46], fibrosis with inflammation of portal tracts and paucity of normal bile ducts.
In addition, in patients with extrahepatic obstruction, proliferation and dilatation of inter
lobular ducts and an increased number of periportal PMNs can be observed. Endoscopic
retrograde cholangiopancreatography (ERCP) is the next choice test for diagnosis, but it
is invasive, for this reason its role is under debate [53]. Transhepatic cholangiography
can be used if ERCP is unsuccessful, but again is invasive. Non-invasive alternatives to
ERCP are: magnetic resonance cholangiopancreatography (MRCP), which is increasingly
used and is useful for excluding other disease and evaluating the biliary system [54].
Transient elastography (FibroScan) has potential as a non-invasive method for detection
of cirrhosis in patients with more advanced liver disease [55].
PSC shares many clinical biochemical and pathologic features with primary biliary cirrhosis,
although PSC, can affect both intrahepatic and extrahepatic ducts. PSC is strongly associated
with inflammatory bowel disease, particularly ulcerative colitis. Due to its major morbidity
and mortality the diagnosis has to be confirmed. At the time of diagnosis, PSC typically in
volves both intra and extrahepatic bile ducts in the majority of cases. The most dismal se
quel of PSC is the development of colangio carcinoma (CC) in 14% of patients (which may
not be demonstrable radiographically with the usual diagnostic methods) [56].
A wide spectrum of disease severity exists, ranging from patients who present with ad
vanced liver disease requiring liver transplantation within a short time to those who remain
47
48
asymptomatic for decades. The natural course of PSC is determined by interindividual vari
ability, the rate of progression and the development of CC, which can occur at any time.
The differential diagnosis has to be established among : autoimmune hepatitis, overlap syn
dromes, infectious hepatitis, other bile duct diseases presenting as acute or chronic cholangi
tis, and biliary strictures, cholangiocarcinoma, gallstones, hepatomegaly and primary biliary
cirrhosis.
Liver biopsy in PSC is only needed to diagnose small-duct PSC or to exclude other diseases
that may be associated with PSC or with similar features and confounding aspects. Liver bi
opsy also may be useful for staging the disease. However, serial liver biopsy in monitoring
the disease is not indicated [57].
Recently some authors have developed the Mayo clinic risk score, a multivariate statistical
survival model, on the basis of the long-term course of the disease in 486 PSC patients seen
at three centers in United States. In this score, the need for liver biopsy has been eliminated.
This scoring system has its advantages; it is non-invasive and was found to be well correlat
ed to actual survival. It also performs better than the Child-Pugh classification for cirrhosis,
which does not predict survival with PSC [58].
7.3.3. Autoimmune Hepatitis (AIH) with overlap variants
Overlap syndromes of AIH are not uncommon but are not well defined. Histology, clinical
and serological indicators imply more than one liver disease at the same time.
The diagnosis of an overlap syndrome relies on the biochemical profile, either cholestatic or
hepatitic in addition to the auto-antibodies pattern and elevated gamma globulins. The his
topathology can show portal inflammation with or without involvement of bile ducts [59].
In adult patients with an overlap of PBC and AIH, which is the the most common, antinu
clear as well as antimitochondrial antibodies are present. Chronic hepatitis C may trigger
autoimmune activation, with concomitant positive autoimmune antibodies. AIH may be as
sociated with Ig G4 autoimmune cholangitis (IAC). In contrast to PSC, IAC-IgG4, has no as
sociated intestinal bowel disease and pancreatitis [60].
The value of a biopsy in liver diseases such as PSC or suspected metastatic disease, which is
characterized by a zonal affection of the liver has to be dealt with individually and complet
ed with other imaging techniques.
Liver biopsy is advisable if diagnostic tests show abnormal liver function results which may
be indicative of many etiologies e.g. nonalcoholic steatohepatitis with strongly elevated anti
nuclear antibodies and abnormal iron studies, or co-infection with HIV and hepatitis C in a
patient with abnormal liver function tests taking hepatotoxic drugs etc.
7.4. Alcohol: Fibrous progression related to alcohol injury
Many patients with ethanol injury show initial scarring around central veins with deli
cate fibrosis along the sinusoids [61]. Eventually, bridging fibrosis connects central veins

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and portal tracts. When cirrhosis is fully developed, most of the native central veins
have been obliterated. Alcoholic cirrhosis is micronodular and the scarring is relatively
uniform throughout the liver. With complete alcohol abstinence, the nodules can regener
ate to a larger size, but the central veins are decreased in number and the nodules may
lack some portal tracts [62].
7.5. Non-Alcoholic Fatty Liver Disease, (NAFLD) and Non-Alcoholic Steatohepatitis,
(NASH)
The histological appearance in these disorders may be very similar to the injury related to
alcohol. In non-alcoholic steatohepatitis, the liver exhibits fat and perivenular sinusoidal col
lagen deposition and may be indistinguishable from alcoholic perivenular fibrosis on histo
logical grounds alone. Clinical correlations are basic for its diagnosis [63].
Sometimes a biopsy shows a pattern which looks like alcoholic hepatitis, but the patient de
nies alcohol use. A differential diagnosis for alcoholic hepatitis has to be done, and non-alco
holic fatty liver disease, (NALDF) and non-alcoholic steatohepatitis, (NASH) should be
considered [63].
For many decades, typical alcoholic hepatitis was often diagnosed with liver biopsy, and
in some patients medical records were completed with somewhat judgmental comments
about their persistent denial of alcohol intake. Now, there are other known causes for Mallo
ry bodies (*) and steatosis found in liver biopsies which, in the past, were classified as alco
hol related liver injury. In retrospect, we now know that many patients with alcoholic
hepatitis were treated unfairly [64].
It is clear that similar patterns of injury can be seen in non-alcoholics, especially in the set
ting of diabetes and obesity, referred to as nonalcoholic steatohepatitis (NASH) or nonal
coholic fatty liver disease (NAFLD). This represents a significant form of chronic liver
disease in both adults and children, with a spectrum ranging from indolent to end-stage
liver disease. It may be an underlying cause of cryptogenic cirrhosis and has been report
ed to recur after a liver transplant. Other conditions associated with NASH include acute
starvation, accelerated weight loss, intestinal bypass, disorders of lipid metabolism, and
various drugs. Careful clinicopathologic correlation is required to determine the cause.
Liver biopsy evaluation allows us to establish the degree of steatosis, inflammation, and
fibrosis stage [65].
Liver steatosis
The diagnosis of liver steatosis has several implications in chronic liver diseases.
Liver steatosis is associated with liver fibrosis progression and a decreased rate of sus
tained viral response in chronic hepatitis C.
Donor liver macrovesicular steatosis is independently associated with graft failure at one
year after liver transplantation.
After major hepatic resection, liver steatosis induces an increased risk of post-operative
complications and elevated risk of death.
49
50
Finally, liver steatosis is the main lesion observed in non-alcoholic fatty liver disease
(NAFLD) which, as a consequence of the worldwide burden of visceral obesity, is now an
important cause of chronic liver disease in western countries.
At present, the histological examination of a liver biopsy continues to be the reference for
evaluating liver steatosis despite its limitations. The procedure is invasive and impaired
by sampling bias, which results in imperfect reproducibility and only allows for a semiquantitative grading of steatosis [66]. The non-invasive diagnosis of liver steatosis is done
by imaging techniques and blood tests, but diagnostic accuracy remains to be validated
and their use in clinical practice has yet to be recommended. Ultrasonography is consid
ered the imaging technique of choice for steatosis screening, but its sensitivity in detecting
fatty liver is only 6094% and is operator dependent. Other techniques, such as computed
tomography, proton magnetic resonance spectroscopy and magnetic resonance imaging
offer high accuracy for quantification of liver fat but have low availability, high cost and
lack standardization [67].
The diagnosis of hepatic steatosis and steatohepatitis or non-alcoholic steatohepatitis
(NASH) is not yet possible without liver biopsy. Therapeutic targets of drug development
are in early stages. As regards the study of factors most likely associated with disease pro
gression, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) has
sponsored the NASH Clinical Research Network (CRN) who has developed a histological
scoring system, which is used for clinical trials for NASH [68].
The histological lesions for the diagnosis of NASH are: zone 3 macrosteatosis, hepatocyte
ballooning and mixed lobular inflammation. Other findings that are common include mildmoderate portal inflammation, acidophil bodies, glycogenated nuclei, lipogranulomas and
perisinusoidal fibrosis. In addition, the following may be present: Mallorys hyaline (*) in
hepatocytes, megamitochondria and mild siderosis.
(*) Mallory bodies or Mallorys hyaline are irregular, rope-like eosinophilic intracytoplasmic
strings that represent aggregates of cytokeratin filaments. The cytokeratins form a filamen
tous support network within the hepatocytes. Cellular damage is due, for example, to etanol
producing hepatocyte ballooning degeneration, which can cause the keratins to misfold and
aggregate. Mallory bodies may be found in alcoholic, nonalcoholic steatohepatitis, and Wil
son disease, cholestatic conditions such as primary biliary cirrhosis (PBC) and with certain
drugs, such as amiodarone. Although the fat and neutrophils can resolve relatively quickly
after alcohol abstinence, hyaline can take up to 6 weeks to disappear [69].
The histological severity of NAFLD is determined by the Non-alcoholic fatty liver disease
Activity Score (NAS) and the Fibrosis Score, developed and validated by the CRN [68]. This
scoring system is very useful for assessing change in clinical trials but it is not meant to re
place a full interpretation of histological findings by a pathologist [70].
Some investigators have observed that there is significant sampling variability and that
the histological lesions of NASH are unevenly distributed throughout the liver parenchy
ma and can lead to substantial misdiagnosis and staging inaccuracies. For example, Rat
ziu et al. reported that on 51 patients with NAFLD who underwent paired biopsies, the

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discordance rate for steatosis would have been missed in 24% of cases if only one biopsy
had been done and a difference of one stage of fibrosis or more was seen in 41% of
paired biopsies [71].
7.6. Liver injury caused by drugs
Drug and toxin induced liver injury is a common cause for abnormal liver tests in humans
[72]. Liver injury related to drugs can be subdivided into intrinsic and idiosyncratic injury.
Intrinsic injury is produced through direct or indirect mechanisms and idiosyncratic injury
may be mediated by hypersensitivity or by metabolic toxic metabolites [73].
Drug induced liver cell injuries have different morphological patterns such as, hepatocellu
lar injury, cholestatic injury, bile duct injury, vascular injury, portal fibrosis, neoplasia or
miscellaneous (pigments and inclusions).
The list of implicated products is very long and in some cases mixed lesions can be
found. Drug signature is a well-known concept which implies that the drugs responsi
ble for the injury can be identified from the different lesions it causes to the liver. For
example, diclofenac and minocyline produce a chronic hepatitis pattern, steatohepatitis
can be induced by amiodarone and tamoxifen, vascular toxicity may be associated with
azathioprine etc. [74].
Histological changes that suggest drug- or toxin-related liver injury are atypical therefore, in
some cases, depending on the findings, it is worth the pathologist asking the clinician specif
ic questions in order to do a differential diagnosis and to identify the drug [75]:
Is the patients blood analysis compatible with hepatitis? Has viral injury been excluded?
What are the patients toxic exposures at work, home, or play?
Has every drug been sought and disclosed?
Granulomas (**) may also be part of the inflammatory reaction in drug injury [76].
If granulomas have been found, have other causes of granulomas been excluded? (see
below) [77]
If significant fatty change is found is there any possibility that it could be related to toxic
ethanol injury?
If an abundance of eosinophils is observed in a liver biopsy, a hypersensitivity reaction is
suspected which may resemble viral hepatitis. Eosinophils may also be present nonspecifi
cally in viral hepatitis, in connective tissue disorders, and in some neoplasms (usually in
Hodgkins disease infiltrates). However, when eosinophils are a striking feature, it is advisa
ble that the clinician search for a drug, a toxin, or even a nutritional supplement (natural
medicines).
If numerous liver cell mitotic figures show up in the liver biopsy, this may suggest that a
short episode of drug exposure is to blame.
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7.6.1. Drug Induced Liver Injury (DILI) examples

The American Association of Reumatology has provided guidelines for monitoring pa
tients receiving Methotrexate therapy as there is a known relation between this treatment
and hepatotoxicity [78].
A few years ago methotrexate was used for treating reumathoid arthritis. Now patients with
psoriasis are also treated with this drug, albeit at a lower dose. Many potentially hepatotoxic
medications, used in such cases are worth investigating [78].
Amoxi-clavulanic acid is one of the examples of a broadly used antibiotic which has
been implicated in liver toxicity. Typically the patient with this toxicity presents with
jaundice. After excluding other causes, such as viral hepatitis, autoimmunity, or other
etiologies, and in presence of a normal biliary tree, a liver biopsy is recommended,
which may show a cholestatic hepatitis pattern. After discontinuation of the drug the
evolution is usually favourable [79].
7.6.2. Granulomas in liver biopsies
Granuloma is defined as an aggregate of histiocytes and can only be diagnosed through his
topathological examination.
Causes of granulomas in the liver: most systemic granulomatous diseases involve the liver
to some extent; tuberculosis and sarcoidosis are the most common causes [80]. Other infec
tious agents include bacteria (brucellosis, nocardiosis, tularemia, Q fever [Coxiella burnetii],
spirochetes), various fungi, protozoa, and viruses (cytomegalovirus, Epstein-Barr virus).
Noninfectious causes in addition to sarcoidosis include PBC, drug reaction, extrahepatic in
flammatory disease, such as chronic inflammatory bowel disease, rheumatoid arthritis), neo
plasms (Hodgkin disease) and foreign substances (talc, mineral oil) .
7.6.3. Can negative stains for fungi and acid-fast bacilli exclude infection in patients with fever of
unknown origin?
Definitely not. Cultures for these organisms are more sensitive than special histological
stains. If infection is a possibility, a core of liver should be submitted with sterile precautions
and without fixative to the microbiology laboratory. In addition, tissue in formalin should
be sent to the pathology laboratory for microscopic sections. A tissue sample may also be
sent for molecular analysis to determine whether an infectious agent is present, depending
on the possibilities [82, 83].
7.6.4. Different types of granulomas useful in determining specific diagnosis
Epithelioid granulomas are nodular aggregates of plump macrophages, often associated
with multinucleated giant cells, lymphocytes, and plasma cells. They are typically seen in
sarcoidosis. The presence of central caseating necrosis suggests tuberculosis.
Fibrin-ring granulomas are formed by a fibrin band encircling a lipid droplet, with associ
ated inflammation. They were first described with Q fever but may also be seen after in

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fection with cytomegalovirus or Epstein-Barr virus as well as with drug (allopurinol)

toxicity and in association with systemic lupus erythematosus.
Lipogranulomas are composed of lipid deposits and vacuolated macrophages. They are
formed in the presence of exogenous or endogenous fat accumulation.
Microgranulomas may be a nonspecific finding, they are usually subtle and composed of
small, round clusters of plump Kupffer cells.
There are many causes of hepatic granulomas, including local irritants, infections, infesta
tions and hypersensitivity to drugs. The constituents of these lesions, depending on the eti
ology and inflammatory cytokines produced include large epithelioid cells, multinucleated
giant cells, varied numbers of mononuclear cells and eosinophils. The causes vary in fre
quency from one country to another. Although the etiology may be determined from the
histological features, from special stains for micro-organisms, from culture of part of the bi
opsy specimen or polymerase chain reaction of the paraffin-embedded specimen, or from
clinical and serological data, the cause of hepatic granulomas remains unknown in one third
of cases. It is likely that approximately one third of granulomatous liver reactions are caused
by drugs, including allopurinol, carbamazepine, procainamide, diphenylhydantoin, quini
dine, isoniazid, and sulphanilamide.
7.7. The role of liver biopsy in infections and pyrexia of unknown origin
Although the usefulness of liver biopsy in the diagnosis of fever of unknown origin is still
controversial, a review of the literature shows that liver biopsy can be effective in confirm
ing histopathological diagnosis and microbiological analysis [83].
Based on the findings of a liver biopsy evaluating Fever of Unknown Origin (FUO), we can
conclude that abnormal liver biopsy is helpful in determining the cause of the FUO. The
most common cause of fever was of an infectious origin. Other causes were neoplastic disor
ders or inflammatory [87].
Liver biopsy was performed after routine studies were negative. Therefore results such as
histoplasmosis and tuberculosis indicate that, despite advances in diagnostic technology,
liver biopsy continues to be useful in the diagnosis of FUO. In endemic areas, histoplasmosis
and tuberculosis should be considered in the differential diagnosis of FUO [85].
7.8. Cirrhosis
Cirrhosis is pathologically defined as a diffuse process in which the normal anatomical lo
bules are replaced by architecturally abnormal nodules separated by fibrous tissue. There
fore, focal scarring, even if significant and associated with nodules, is not cirrhosis because
the process is not diffuse [86]. In the past the description of a liver as cirrhotic implied an
ominous prognosis in a patient with liver disease. In chronic hepatitis, the most important
goal is to delay or to stop the development of cirrhosis. Nowadays, treatments to prevent its
progression are available. At present there are many known stages as opposed to before
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when there only one was considered, G Garcia-Tsao in the article In search of a pathophy
siological classification of cirrhosis. [86].
7.8.1. Fibrosis progression
One of the most crucial developments is the reformulation of the concept of cirrhosis from a
static to a dynamic process. This concept is likely to be even better defined in the future.
As fibrotic scars advance and extend the normal architecture changes and nodules are
formed [88]. Moreover, the angiogenic process that naturally accompanies scar formation
permits the creation of abnormal channels between central hepatic veins and portal ves
sels, resulting in the shunting of blood around the regenerating parenchyma. Normal
vascular structures, along with sinusoidal channels, may be obliterated, leading to portal
hypertension. Some authors describe cirrhosis as a vascular disease [89]. Clinical conse
quences of cirrhosis result from the decreased ability of the parenchyma to synthesize
clotting factors and other substances combined with the complications related to portal
hypertension [90].
Knowledge on the level of fibrotic progression between normal histology to cirrhosis has
considerable prognostic weight. Patients with bridging fibrosis on biopsy are much closer to
end-stage liver disease than those with minimal or no fibrosis. Fibrosis is not an autono
mous feature, but rather a tissue progressive lesion resulting from other pathologic mecha
nisms such as inflammatory, degenerative or dystrophic processes [91].
The first transition in this process occurs between the normal, non-fibrotic state and the ex
pansion of the portal area by fibrosis, to the extension of short, incomplete septations
around the portal area, change that gives to the portal areas an irregular stellate shape.
In the next transition, development of bridges between vascular structures, portal-portal
bridging fibrosis and portal-central bridging, occur. Gradually, more and more bridges are
formed, accompanied by distortion of the architecture due to hepatocellular regeneration
and contraction of fibrotic scars. When these changes diffusely involve the biopsy, it is clas
sified as cirrhosis [92].
Progressive fibrosis leads to cirrhosis and it is now known that cirrhosis can be reversible.
There was a lot of controversy surrounding this issue a few years ago [93]. For patients in a
precirrhotic stage of fibrosis, liver biopsy remains the gold standard of assessment. Prior to
1995, there was no published system which subdivided advanced stages of cirrhosis. Only
the Ishak modification of the Histologic Activity Index (HAI) subdivided cirrhosis into three
categories [94].
Nowadays, since Garcia-Tsao et al.reported compensated and decompensated phases in the
clinical evolution of liver cirrhosis, many prophylactic measures and controls have been im
plemented in order to improve survival and quality of life [87]. Cirrhosis is usually clinically
evident. Once the pathologic stage of cirrhosis has been reached, clinical scales such as the
Child-Pugh score have to be used because they represent the prognosis and the staging of

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the liver disease better [95]. The present debate questioning the need for liver biopsy versus
non invasive tests will be discussed below.
7.8.2. A needle biopsy specimen does not always permit the diagnosis of cirrhosis
Micronodular cirrhosis (nodules of 3 mm or less), which may develop as a result of ethanol
injury, biliary tract disease, or hemochromatosis, is usually uniform throughout the liver,
and nodules may be identified on a needle specimen. However, macronodular cirrhosis
(nodules greater than 3 mm), due most commonly to chronic viral hepatitis, constitutes a
less uniform pattern [96].
7.9. Hepatocellular Carcinoma (HCC) and other benign or malignant focal lesions: The
role of Fine Needle Aspiration Biopsy (FNAB) [97]
Indications of liver biopsy with regards to diffused or local lesions
Liver biopsy is useful for diagnosis of a diffused disease and guided liver biopsy remains
essential for the diagnosis of localized lesions.
7.9.1. Fine needle aspiration biopsy (FNAB)
This technique has a crucial role in the evaluation of focal liver lesions or localized lesions.
Liver tumors appear as nodular or localized lesions which can be malignant or non-malig
nant and can be either primary from the liver or metastasic. If clinical, biochemical and radi
ologic findings are inconclusive, some phases of the diagnostic process may require a liver
biopsy in order to establish the diagnosis and their staging and management [99].
Malignant lesions. Hepatocellular carcinoma (HCC), the most frequent malignant liver
cancer, is usually discovered during screening programs in cirrhotic patients. Regarding
treatment, the only curative option is surgery, both limited hepatectomy of the tumor or liv
er transplant in very select cases [100].
In liver lesions with typically recognized features of HCC, defined by using advanced radio
logical methods, liver biopsy has no place. However, a liver biopsy will be performed in pa
tients with atypical liver tumors suggestive of a possible colangiocarcinoma. These cases
require another form of therapy and the prognosis is worse [99].
Besides, when surgery is indicated in a patient with suspected liver cirrhosis, a liver biopsy
has to be performed in the non-neoplasic liver. Pathological diagnosis may help to asses the
functional capacity, specific prognosis and whether surgery could be performed.
Metastasis of the liver with an unknown primary tumor should be biopsied to obtain infor
mation of the primary tumor in order to determine therapy.
Concern has been expressed about the risk of spreading malignant cells via the needle tract,
but this rarely occurs when using needles with a diameter of less than 1.3 mm, which also
minimizes the risk of bleeding. The procedure is simple, safe and painless [101].
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7.9.2. Non-malignant lesions

In cases of Hemangioma or Focal Nodular Hyperplasia (FNH), diagnosed and confirmed by
radiology, biopsy is usually not necessary.
FNH and hepatic adenoma are benign tumors and are less frequently observed than HCC.
Their diagnosis is done using imaging techniques (ultrasound or helicoidal scanner). How
ever, differential diagnosis is necessary because, although FNH only requires radiological
follow-up, in some cases, higher risk circumstances have been recognized and surgery is
recommended [102].
7.9.3. Most prevalent mass lesions [102, 103]
Benign: cysts, hemangioma, adenoma, liver abscess (amebic or pyogenic), focal nodular
hyperplasia, fatty infiltration, rare primary liver neoplasms.
Malignant: hepatocellular carcinoma, cholangiocarcinoma, metastatic, rare primary liver
neoplasms, rare primary bile duct neoplasms.
7.10. New evolving fields for liver biopsy: Liver transplantation, Bone marrow
transplantation, Living donors and Morbid obesity
7.10.1. Liver transplantation
With regards to liver transplantation, liver biopsy remains very useful in the management of
transplanted patients. In this clinical situation, if a rejection is suspected and other complica
tions have been ruled out, a guided biopsy will be performed. This procedure can be of
great value in order to confirm the specific diagnosis and to indicate treatment [104].
In the first few weeks and months after transplantation, the major causes of abnormal liver
tests include preservation injury, acute rejection, opportunistic infections (e.g., cytomegalo
virus, hepatitis), vascular compromise, and/or biliary stricture. Of these, acute allograft re
jection is the most common and results from direct alloantigenic stimulation of recipient T
cells by donor dendritic cells (antigen-presenting cells). The effector T cells can then prefer
entially injure biliary epithelial cells of both interlobular and septal bile ducts as well as en
dothelial cells of intrahepatic arteries and veins.
The main histological features of acute rejection Acute rejection is characterized by an infiltration
of mixed, predominantly mononuclear cells within portal tracts. The inflammatory infil
trates include lymphocytes, macrophages, plasma cells, polymorphonuclear neutrophils and
eosinophils. The inflammatory cells typically infiltrate the bile duct epithelium and are asso
ciated with bile duct damage. Subendothelial inflammation (endothelialitis), which may in
volve both portal and central veins, is also a typical feature. The most common grading
system is the Banff schema, a consensus document proposed by an international panel of
pathologists and liver transplant physicians [105]. Criteria helping to distinguish recurrent
hepatitis C after transplantation from allograft rejection; Hepatitis C (HCV) recurs in virtual
ly all patients transplanted for that disease.

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Distinguishing recurrent hepatitis from acute allograft rejection, which can overlap, is diffi
cult. There are usually three main phases to recurrent HCV:
Graft reinfection (from 0 to 3 months post-transplant). HCV-related inflammation is rare
ly seen at this time. Liver biopsies may show mild lobular disarray, few necrotic hepato
cytes (acidophil bodies), and fatty change.
Established graft infection (from 3 to 6 months), acute hepatitis including ballooning de
generation of hepatocytes, acidophil bodes, and Kupffer cell prominence can be observed.
Varying degrees of portal tract inflammation may also be present.
Progressive liver damage (after 6 months), features related to chronic HCV infection such
as, mononuclear portal infiltrates and interface hepatitis are observed. Bile duct damage,
although mild, may occur, and granulomas may be detected. Up to half of patients will
have histological evidence after 1 year.
The role of liver biopsy in the evaluation of abnormal liver tests after the first year post transplanta
tion Common causes after the first year include acute rejection, opportunistic infection, re
current viral hepatitis, chronic rejection, steatohepatitis, or recurrent diseases. Chronic
rejection occurs as a consequence of repeated episodes of acute rejection that are unrespon
sive to immunosuppression. The main histological abnormalities are loss of small bile ducts
(ductopenic rejection) and/or obliterative vasculopathy (affecting large and medium-sized
arteries). Unlike acute allograft rejection, the degree of bile duct damage is typically out of
proportion to the degree of inflammation.
Complications of liver transplantation are not limited to acute and chronic rejection and re
currence of original disease, but include surgical complications, most commonly hepatic ar
tery occlusion, infections, and development of de novo malignancies. In the early post
transplantation period preservation injury, damage to the graft during harvesting and im
plantation, may lead to significant graft dysfunction. In post-perfusion biopsies, heavy neu
trophilic infiltrate and hepatocyte necrosis may be predictive of initial poor graft function.
Ischemic complications, such as hepatic artery thrombosis, are one of the most serious com
plications and may lead to early graft loss or biliary stricture. In these patients liver biopsy is
usually not performed.
Infectious complications that generally occur after transplantation, cytomegalovirus(CMV)
for example, remains common and is frequently associated with parenchymal microabscess
es which are found in the liver biopsy of CMV patients.
7.10.2. Bone marrow transplantation
A liver biopsy is effective in the evaluation of a bone marrow transplant recipient with ele
vated liver tests [106]. Known complications of bone marrow transplantation include venoocclusive disease (VOD) and graft-versus-host disease (GVHD). A biopsy is necessary to
diagnose VOD. It develops within 1 to 4 weeks after transplantation and is characterized by
occlusion of central veins, sinusoidal fibrosis, and pericentral hepatocyte necrosis. Acute
GVHD develops within 6 weeks after transplantation and affects the skin, gastrointestinal
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tract, and liver. It is characterized by degenerative bile duct lesions with some degree of
mononuclear inflammation. Cholestasis may be present. Chronic GVHD is a multiorgan
process that develops 80 to 400 days after transplantation and is often preceded by acute
GVHD. The changes in the liver are similar to those in acute disease, but the ducts show
more prominent changes and are likely to be reduced in number or destroyed. A prominent
periportal mononuclear infiltrate, or even piecemeal necrosis, may be seen.
7.10.3. Liver transplant living donor
Liver biopsies detect silent donor disease in potential living liver donors, especially patients
suffering subclinical non-alcoholic fatty liver disease (NAFLD). The contribution of liver bi
opsy or even the need to perform this, when a potential donor is being evaluated is a contro
versial issue [107]. In the University of Pittsburgh Medical Center a retrospective study of the
histopathologic examination and diagnoses of 284 patients, who were evaluated as living do
nors from 2001 to 2005 was carried out. Hepatic histology was correlated with liver injury
tests and with demographic characteristics in an otherwise normal healthy population. A mi
nority (n=119; 42%) of biopsies from this population of 143 males/141 females (average
age=36.8years; mean BMI=26.6) were completely normal. The remainder showed steatosis
(n=107; 37%), steatohepatitis (n=44; 15%), or unexplained low-grade/early stage chronic hepa
titis, primary biliary cirrhosis, or nodular regenerative hyperplasia (n=16; 6%). Biopsy find
ings disqualified 29/56 donors, negative factors were: obesity, age and liver iron content,
contributing to NAFLD pathogenesis. The conclusion was that liver biopsy provides valuable
information about otherwise undetectable liver disease in potential liver donors.
7.10.4. Morbid obesity
About 90 per cent of morbidly obese patients show histological abnormalities of the liver.
Morbid obesity may lead to severe disease showing steatosis, ballooning degeneration,
lobular inflammation and fibrosis in the study of liver biopsy. These features are similar
to the lesions observed in alcoholic hepatitis and may end in cirrhosis and liver failure.
Many factors such as, alcohol, drugs, diabetes, viruses, can contribute to progressive liver
damage. The development of severe fatty liver disease may be asymptomatic showing a
poor correlation with liver function tests. It has been reported that after bypass surgery,
weight loss is accompanied by improvement in fatty change and the liver function tests
are normal.
Histopathologic findings in the liver of 160 patients who were undergoing laparoscopic gas
tric bypass or gastric banding for morbid obesity, were recorded, also clinical data (gender,
age, BMI and associated diseases) and laboratory evaluation were obtained [108].The diag
nosis obtained were : 63 non-nonalcoholic steatohepatitis (non-NASH), 54 NASH, 26 chronic
hepatitis B (CHB), 15 alcoholic steatohepatitis and NASH, and 2 chronic hepatitis C
(CHC).The coexistence of clinical and histological features of steatohepatitis with another
chronic liver disease may reflect the biological significance of the chronic inflammatory con
dition in the obese population, which requires further investigation.

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8. Non-invasive tests for liver disease and assessment of fibrosis and

cirrhosis
Limitations of biopsy have led clinical investigators to study alternative methods to in
vestigate liver disease especially for the assessment of liver fibrosis. Since fibrosis is of
sufficient importance in chronic liver disease and because it progresses to cirrhosis it is
frequently used as the outcome and prognostic variable in clinical studies. Hepatic fib
rosis is currently viewed as a dynamic process that may regress after successful treat
ment of chronic liver diseases. Serum markers, such as non-invasive markers, offer an
attractive alternative. They are objective, allowing a dynamic calibration of fibrosis, can
be performed repeatedly, are more cost effective and many of them are performed as a
routine analysis [109].
Indirect markers of liver fibrosis: among them, the AST to Platelet Ratio Index (APRI),
combines aspartate amiotransferase (AST) with platelet count. It was used in several
studies conducted in cohorts of patients with hepatitis C and showed a rather good di
agnostic performance and reproducibility, [110] particularly for cirrhosis. Forns and col
leagues reported a fibrosis index (Forns index) based on platelet count, -glutamyl
transferase (GGT), and cholesterol levels [111]. It is rather good for excluding significant
fibrosis, but only average for diagnosing significant fibrosis. However, one important
limit of both APRI and Forns indexes are that they leave almost half of the patients
unclassified.
Another widely investigated combination set of noninvasive markers of liver fibrosis is the
Fibrotest; a combination of five blood tests based on a mathematical formula: GGT, biliru
bin, haptoglobin, apolipoprotein A1, alfa2 macroglobulin adjusted for gender and age. Ac
cording to the investigators, it could exclude cases with significant fibrosis (METAVIR > F2),
having 100% of negative predictive value, and more than 90% positive predictive value, us
ing liver biopsy as a reference [112].
8.1. Elastography (FibroScan)
Another noninvasive method for the assessment of liver fibrosis is elastography (Fibro
Scan) [113].
FibroScan device (EchoSens, Paris, France) uses a mild-amplitude, low frequency vibration
transmitted through the liver. It induces an elastic shear wave that is detected by pulse-echo
ultrasonography as the wave propagates through the organ. The velocity of the wave corre
lates with tissue stiffness which correlates well with the degree of fibrosis.
This device is in widespread use in many parts of the world, but is not yet approved in the
United States.
Most of the studies have been conducted on patients with chronic hepatitis C but a few
studies have also covered fibrosis and cirrhosis due to other etiologies.
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This technique, however, has its limitations: it uses expensive equipment, and has decreased
accuracy in obese patients and in patients with ascites. Elastography results are not valid in
presence of hepatic steatosis, cholestasis, necroinflammation, or portal hypertension. The
patients age and levels of aminotransferases need to be taken into account when interpret
ing results of liver stiffness [114].
IN FAVOUR of LIVER BIOPSY

Chronic HCV hepatitis
AGAINST OF LIVER BIOPSY
In selected indications for grading and Not necessary for diagnosis. Possible
staging
use of non-invasive methods in followup controls
Chronic HBV hepatitis
Grading and staging advisable before Not necessary for diagnosis. Possible
starting treatment
use of non-invasive methods in followup controls
Non-alcoholic steatohepatitis (NASH) NASH is a always an histopathological Assessment of fibrosis possible with
Alcoholic steatohepatitis (ASH)
Autoimmune Hepatitis
Primary Biliary Cirrhosis (PBC)
Hemochromatosis
diagnosis
non-invasive methods
ASH is a histopathological diagnosis.
Assessment of fibrosis possible with
But in alcoholic acute hepatitis liver
non-invasive methods,( in abstinent
biopsy usually is not performed
patients)
For diagnosis and staging liver biopsy is Non validated methods yet for nonneeded
invasive assessment of fibrosis
Not needed in typical mild cases
Possible non-invasive assessment of
without biliary duct damage
fibrosis
In general, liver biopsy performed for
Non validated methods for non-
diagnosis and staging, and iron
invasive assessment of fibrosis
content in the liver

Wilson disease
For diagnosis and staging and copper Non other options

content in the liver
Table 5. Indication for liver biopsy in different chronic liver diseases in the era of non-invasive methods
In spite of that, elastography is complementary as the combination of noninvasive markers

and elastography improves the overall accuracy. In one of the metanalysis, for significant
fibrosis, the area under the ROC for Fibrotest and FibroScan were 0.81 (95% CI 0.78-84) and
0.83 (0.03-1.00), respectively [115].

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Fibrotest, and elastography (Fibroscan) as first line estimates of fibrosis in patients with
chronic hepatitis C are recommended and liver biopsy will probably be indicated only as a
second line diagnosis and reserved for cases of discordance or non-interpretability [112].
Some authors conclude that elastography appears reliable to detect significant fibrosis
and cirrhosis in patients with chronic hepatitis C, besides it may turn out to be a valua
ble diagnostic procedure and follow-up of patients with chronic liver diseases of differ
ent causes [115].
8.2. Liver biopsy: Consensus among pathologists?
It is crucial that biopsy interpretation be done by experienced liver pathologists. Pathol
ogists have tried to define the features (including length and number of complete por
tal tracts) of an adequate liver biopsy sample able to correctly assess the classification
of liver fibrosis. Some authors have recommended big samples of 1 to 4 cm in length
containing at least 11 complete portal tracts, which could be more reliable for adequate
grading and staging [116, 117]
Feature
Effect on response to therapy
Effect on natural history
Fibrosis
Reduces response
Presence implies progression
Inflammation
No effect
Related to increased amount of current

fibrosis and increased rate of
progression
Steatosis
Reduces response

fibrosis, unclear effect on progression
Iron accumulation
Unclear effect

fibrosis, unclear effect on prospective
rate
(adapt from Semin Liver Dis 2005)

Table 6. Relevance of histological features of chronic hepatitis C to disease progression and therapeutic response
Many intraobserver and interobserver variations have been estimated in the assessment
of features, classification, and scoring of liver biopsy assessment. One study reported dif
ferences in the evaluation of liver biopsies in chronic viral hepatitis C among 10 patholo
gists specializing in liver diseases. These pathologists independently reviewed 30 liver
biopsy specimens of viral hepatitis C and completed a histological form for each of the
specimens. Five pairs of pathologists were then randomly designated and they inde
pendently reviewed the biopsy specimens and filled out a new coding form. The inter
observer variation was calculated for each item among the 10 individuals and then
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among the five pairs. Five features showed an almost perfect or a substantial degree of
concordance among the 10 observers (cirrhosis, fibrosis, fibrosis grading by Knodell in
dex, steatosis and portal lymphoid aggregates). The 17 other indicators showed a weak
er concordance. Five items had a higher concordance when viewed by a pair of
pathologists than when studied by only one pathologist (steatosis, periportal necrosis
grading by Knodell index, lobular necrosis grading by Knodell index, centrilobular fibro
sis, and ductular proliferation). This study reveals that certain features of major impor
tance in assessing disease activity show significant observer variation. The acceptable
degree of concordance was related mainly to the fibrosis score, whereas other numerical
items displayed substantial observer variations. Simultaneous observation by two pathol
ogists increased the reproducibility of numerical scoring and of certain viral hepatitis C
lesions. A classification of chronic hepatitis C based on dissociated semiquantitative as
sessment of necroinflammatory lesions and fibrosis offers more reproducibility than the
use of a global numerical index [107].
As a single percutaneous liver biopsy yields only a minute percentage (1 50 000 or 0.002%)
of the total hepatic tissue, paired biopsies have been evaluated in several published studies,
especially for assessing steatosis and NASH. For quantification, better references are re
quired, such as imaging techniques or morphometry, which determines the area of steatosis
on liver biopsy.
In fact, as liver steatosis is not homogeneous, classical optical examination of a liver biopsy
by a pathologist for measuring liver steatosis by the determination of the percentage of hep
atocytes containing lipid vesicles is highly subjective, and steatosis grading corresponds on
ly to a semiquantitative scale [68].
The role of the liver biopsy in disease management is evolving nowadays and has to be re
considered given the modern pathologic assessment of liver biopsy. Pathologists have made
progress in the interpretation of liver biopsies and in processing the information in a concise
and scientific way available to clinicians. After evaluating the disease state and interpreting
the tests results, the clinician in charge of the patient should consider the individual patient
when making recommendations with regards to treatment.
Role of the liver biopsy in personalized medicine
The liver biopsy specimen aims to obtain a valuable material for the assessment of fibrosis
and cirrhosis. Despite limitations related to sampling and interpretation, histological exami
nation remains the best standard for staging and diagnosing chronic liver diseases. Its indi
cations are decreasing because new therapeutical options for chronic viral hepatitis have
improved [118]. Moreover, new non-invasive tests have been developed and their use may
increase in the future, especially for long term management [119] (Table 7).
All invasive procedures involve risks, consequently the benefits of obtaining liver for histol
ogy should always be weighed against the possible morbidity of the procedure. The deci
sion to indicate a liver biopsy has to be taken depending on the centers facilities and the
availability of experienced liver pathologists to interpret the biopsy.

http://dx.doi.org/10.5772/53120
Ethics related to liver biopsy mainly include issues on the indications, information on poten
tial risks and benefits and validity of available alternative options. Patients should be ade
quately informed and participate in the decisions for liver biopsy and treatment [120].
Advantages
Liver biopsy
Transient elastography
- Direct measure of liver fibrosis
- Non-invasive
- Well established staging system
- Easy to repeat
- Assessment of architectural
- No risk to patient
disturbances related to liver fibrosis
- Performed in the outpatient clinic
- Evaluation of associated lesions
- Results available immediately
(inflammation, steatosis, iron, alcohol) - Reproducible

- Highly performance for detecting
cirrhosis
Disadvantages
- Invasive and painful
- Unable to discriminate between
- Difficult to repeat
intermediate stages of fibrosis
- Potentially severe complications
- False positive in case of acute
- Contraindicated if ascites,
hepatitis, extrahepatic cholestasis and
coagulopathy etc.
congestive heart failure
- Sampling and interobserver variability

(adapt L Castera & M Pinzani, Gut 2010)
Table 7. Use of liver biopsy in clinical practice. Respective advantages and disadvantages of liver biopsy and transient
elastography for assessing fibrosis in chronic liver disease
12. Conclusions
What will be the real impact of Liver Biopsy now and in the near future in the era of person
alized medicine?
1.
The practice of liver biopsy will remain as an important component in the evaluation of
liver disease. However, the value of liver biopsy should be contemplated as a comple
mentary tool in modern medicine because of the presence of new non-invasive diagnos
tic measures, better prognostication methods and more advances in imaging
techniques.
2.
Non invasive tests such as Fibroscan, or similar, adding serum markers will be increas
ingly used to identify the amount of fibrosis, and will spare, in most patients, the per
formance of a liver biopsy.
3.
Liver biopsy provides information that is used in conjunction with other data to inform
and to guide therapy. The team that joins pathologists, clinicians, radiologists and other
specialists meets in order to make clinico-pathological correlations. New classifications
incorporating clinical data in the histological dictamen will be implemented.
63
64
4.
Therapies, etiology, pathogenesis, cellular and molecular mechanisms, changes in tissue

architecture and invasive (HVPG) and noninvasive diagnostic approaches, should be
added to the liver biopsy information.
5.
In the liver transplant field, liver biopsy has allowed many scientific advances and in
most of these patients liver biopsy will continue to be mandatory for their management.
6.
Patients seeking a second opinion or who are referred to a tertiary care center, will re
quire a deep review of previous obtained specimens in order to confirm and to plan
their management.
7.
Since chronic viral hepatitis is a prevalent disease in the general population, the number
of liver biopsies will be limited in the next years because it is costly and aggressive so
validated non-invasive methods will be favoured.
8.
Nevertheless, some questions surrounding non-invasive markers will remain. Non-in

vasive markers have been validated against the biopsy, and the overall accuracy of bi
opsy is only 80%, it is probably statistically impossible for a marker to perform any
better than a biopsy.
9.
Increasing research on hepatic fibrosis, diagnoses and therapy, is ongoing so valuable

results of predicting changes in fibrosis content over time have to be followed by histo
logical liver assessment. Moreover, considering the possible regression of cirrhosis, now
clinicians and pathologists have become more interested in studying histological fea
tures of tissue repair and fibrosis regression in the liver.
10. The number of liver biopsy will be sparing in common patients but it will play a crucial
role in research; for example studying rare diseases, stem cells and genetic disorders.
Moreover, its role is evolving in many research fields such as obesity, bone marrow
transplant, and oncology.
Acknowledgements
I would like to thank Aisling Dowd, for her help while preparing the manuscript
Author details
Chronic Hepatitis Coordinator Program, Liver Transplant Unit, Hospital Universitari de
Bellvitge, LHospitalet de Llobregat, Barcelona, Spain

http://dx.doi.org/10.5772/53120
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Section 2
Clinical Practice of Liver Biopsy
Chapter 4
Safety and Reliability Percutaneous Liver Biopsy

Procedure in Children with Chronic Liver Diseases
Anna Mania, Pawe Kemnitz,
Magdalena Figlerowicz, Aldona Woniak and
Wojciech Suewski
http://dx.doi.org/10.5772/52619
1. Introduction
Liver biopsy remains a golden standard in the evaluation of various liver diseases. It is one
of the most specific test allowing to assess the severity of various liver diseases. Clinical
evaluation may be inadequate as chronic liver diseases could be asymptomatic for a long pe
riod of time. The routinely used laboratory test may be irrelevant, as diffuse changes may
possibly be present in the liver in spite of liver function test being within reference values.
Percutaneous biopsy allows to obtain a tissue specimen suitable for pathological assessment.
Liver biopsy is an important procedure in diagnosing liver diseases in infants and children
as it often provides diagnostic information not possible to obtain by other methods. There
fore, liver biopsy is considered to be a golden standard in the diagnostics and follow-up of
the patients with chronic diffuse hepatopathies. The role of the liver biopsy is to confirm the
diagnosis of chronic hepatitis, assess the necroinflamatory activity (grading) and the severi
ty of fibrosis (staging), confirm the presence of cirrhosis. Other hepatopathies may be ex
cluded as well as associated diseases using this method [1].
The size of liver sample varies from 1 to 4 cm in length and 1.2 to 1.8 mm in diameter. Biop
sy specimen represents 1/50,000 of the total mass of the liver, therefore the procedure carries
the risk of sampling error. The specimen should be sufficient in length (2-2.5 cm) and num
ber of portal spaces (at least 11). The fragmentation of the specimen should be avoided [2].
Liver assessment is also affected by an interpretative error and intraobserver variability of
histological interpretation. Moreover, liver biopsy is an invasive procedure carrying the risk
76
of certain complications including pain, bleeding, pneumothorax, puncture of bile ducts or

the gall bladder.
Repeating samples in different time intervals are useful in monitoring the efficacy of treat
ments. Many patients are, however, reluctant to experience repeated biopsies, which limits
the ability to monitor disease progression and treatment effects. [3].
Due to the limitations of the procedure many non-invasive techniques were developed such
as single serological markers, panels of different markers, imaging techniques and elastogra
phy [4]. None of the non-invasive methods is suitable and reliable enough to entirely substi
tute the liver biopsy. Non-invasive techniques are very helpful in the detection of severe
lesions. However, results obtained from patients with intermediate lesions very often over
lap between different categories of staging. Nevertheless non-invasive methods are useful in
situations where contraindications to liver biopsy do not allow to perform the procedure.
Liver biopsy can be percutaneous, transjugular or laparoscopic. Percutaneous liver biopsy
can be blind, ultrasound- guided or ultrasound assisted. Various approaches differ in the
number of potential complications and require various equipment. Ultrasound guidance al
lows safer intercostal approach and may be useful in the evaluation of relative position of
the liver, gall bladder, kidneys and lungs. The technique reduces the risk of hemothorax and
pneumothorax and puncture of the gall bladder.
The aim of this study was to evaluate safety and reliability of the liver biopsy in children in
relation to obtained results and potential complications.
2. Material and methods

Seventy five cases of percutaneous liver biopsies carried between 2005-2012 were analyzed.
The biopsies were performed in children aged 4-17 years (mean 15.302.35 years). Study
group included 26 girls, 49 boys. Procedures were done due to chronic hepatitis C (CHC)
44 cases, chronic hepatitis B (CHB) 16 cases, autoimmune hepatitis (AIH) 3 cases, hepati
tis/hepatomegaly of unknown origin (HUO) 12 cases, non-alcoholic fatty liver disease
(NAFLD) 2 cases. Number of the procedures performed in the following years has been
presented in Figure 1.
Written informed consent was obtained from the parents and patients aged 16 years and
over according to Polish law regulations. Before the procedure children were clinically eval
uated and blood samples were taken for standard hematological and clinical chemistry anal
ysis. Children with coagulopathies and thrombocytopenia below 80,000/mm3 were excluded
from the procedure. All children underwent abdominal ultrasound performed the day be
fore the procedure to exclude potential hemangiomas and malposition of the organs. All
children were managed by Menghini procedure in sedation. Children aged less than 5 years
received general anesthesia. 36 biopsies were ultrasound guided directly prior to the proce
dure (performed by the operator), 39 biopsies were blind. The ultrasound prior to the biopsy
was performed to identify the intercostal space and to avoid accidental puncture of the gall
Safety and Reliability Percutaneous Liver Biopsy Procedure in Children with Chronic Liver Diseases
http://dx.doi.org/10.5772/52619
bladder, the lung, right kidney and large vessels. Immediately after the procedure ultra
sound examination was performed searching for potential complications such as accidental
puncture or bleeding. In the case of blind biopsied ultrasound examination was performed
by radiologists in situations where complications were suspected basing on clinical symp
toms. All patients were monitored 24 hours after the procedure in the department for vital
signs, pain and other consequences.
Histological evaluation was performed using Ishak scoring system for grading and staging.
Categorical variables were compared using Fishers exact test or chi-square test were appro
priate. Result with p value <0.05 were considered statistically significant.
Figure 1. Number of liver biopsy performed in the Department of Infectious Diseases and Child Neurology due to vari
ous reasons in years 2005-2012 (until July) CHC- chronic hepatitis C, CHB chronic hepatitis B, NAFLD non-alcoholic
fatty liver disease, HUO hepatitis/hepatomegaly of unknown origin, AIH autoimmune hepatitis
3. Results
Liver samples were obtained in all children. Adequate sample size was not obtained in the
case of 5 children - 2 samples were to short and did not contain the adequate number of por
tal spaces, one sample was fragmented. Four inadequate samples resulted from the blind
liver biopsy and 1 was obtained by the ultrasound guided procedure (p=0.21). No significant
adverse events were observed. No clinical signs of hemorrhage, no cases of pneumothorax,
puncture of the gallbladder nor severe infections were observed. Larger bile ducts were
punctured in 4 cases all undergoing blind procedure (p=0.07). 12 patient were complaining
on pain in the right upper quadrant of the abdomen following the procedure that required
more intensive analgesics 3 undergoing ultrasound guided procedure, 9 having blind liver
77
78
biopsy done (p=0.07). Pain was mild to moderate and resolved after analgesics. There were
no deaths following the procedure in both groups of children.
Results from pathological assessment were presented in Table 1. The majority of children
underwent liver biopsy due to CHC. Remaining indications were CHB, AIH, NAFLD, HUO.
In patients with viral hepatitis grading and staging assessed according to Ishak scoring sys
tem was usually mild to moderate. Nevertheless, severe lesions were also present in some
patients. Figure 2 and Figure 3.show examples of inflammatory changes and portal fibrosis
in various patients with CHC. In patients with AIH and NAFLD diagnosis was confirmed
by pathological assessment. Ten of HUO patients gained diagnosis either of metabolic disor
ders or NAFLD thanks to pathological evaluation. Normal liver histology was described in 2
patients.
Figure 2. Liver biopsy specimen of the patient with CHC where inflammatory infiltrates cross lamina basalis of the lo
buli (thin arrows) and intralobular focus of inflammatory infiltrate (thick arrow).Staining: hematoxylin+eosine. Magni
fication 40x
Fifteen children with viral hepatitis underwent repeated procedures allowing to assess the
progression of lesions in time. In 9 of them the progression of lesions was described, 6 had
similar results in both biopsies.
http://dx.doi.org/10.5772/52619
Indication for the biopsy
Result
Number of children
Grading
CHC - 44
23
11
Staging
0
19
17
Grading
CHB - 16
NAFLD - 2
HUO - 12
AIH - 3
Staging
1
Steatosis
Steatohepatitis
Metabolic disorders
Nonalcoholic steatohepatitis
Wilson disease
Normal liver histology
Autoimmune hepatitis
Table 1. Histological assessment of the liver biopsy specimens performed in 75 children. CHC- chronic hepatitis C, CHB
chronic hepatitis B, NAFLD non-alcoholic fatty liver disease, HUO hepatitis/hepatomegaly of unknown origin, AIH
autoimmune hepatitis
4. Discussion
Studies describing the safety of liver biopsy performed on larger cohorts of patients seem to
prove that the procedure results in more complications in children than in adults [5]. Never
theless, Lebensztejn et al. described the group of 250 pediatric patients undergoing blind
procedure with serious complications as internal hemorrhage and puncture of the gallblad
der occurring in 3 children [6].
79
80
Figure 3. Syrius red staining of the liver specimen of CHC patient with present fibrosis in dilated portal space (thick
arrow) and porto-portal bridging. Magnification 40x.
Number of biopsies in the current study was lower, however even the number of mild com
plications was relatively low. Moreover, no serious adverse events were noted among chil
dren from the study group. Noted complications included puncture of larger bile ducts and
pain after the procedure. Although the results were not statistically significant, both prob
lems were more frequent in children undergoing blind liver biopsy. Ultrasound assistance
during the whole procedure was found to reduce the number of potential consequences [7].
Thus, ultrasound guidance even performed right before and after the biopsy makes the
whole procedure safer. Since the majority of complications occur within first hours after the
liver biopsy all children were monitored for 24 hours after the procedure as inpatients. Al
though hospitalization increases the costs of the procedure, monitoring enables quick re
sponse to encountered complications and prompt treatment, if necessary. Another issue is
general anesthesia performed in small children in order to obtain liver sample. Although
costly, general anesthesia decreases fear, pain and enables to perform the procedure in safe
circumstances, reducing the risk of hemorrhage caused by lack of cooperation from the pa
tient side.
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The majority of children underwent the liver biopsy due to chronic viral hepatitis mostly
CHC. Histological assessment was not necessary to establish diagnosis since it is usually
based on blood tests. However, information regarding grading and staging was essential for
treatment decisions since the length of treatment may vary depending on the severity of le
sions. In patients with CHB decisions regarding the initiation of the treatment may depend
on the presence of lesions in the liver tissue [8]. In both types of chronic viral hepatitis pa
tient with liver cirrhosis requires different approach than the child with mild lesions in the
liver. In children with AIH the diagnosis was confirmed by the detection of specific inflam
matory cells in the liver tissue. Although the number of NAFLD was small, the procedure
distinguished between simple steatosis and steatohepatitis. Patients who underwent the
procedure due to HUO benefited from diagnosis in 10/12 children. Metabolic disorders were
detected in 3 patients and steatosis was detected in 6 children, 1 child was found to have
Wilsons disease. Normal liver histology found in the specimens from the following 2 chil
dren with HOU raises questions regarding indications to the liver biopsy. The decision con
cerning the procedure was always carefully made basing on clinical and laboratory findings.
Obtained results may be a consequence of the limitations of the procedure regarding sample
size and sample error related to the site of the biopsy. Diffuse liver diseases are hardly ever
evenly distributed in the organ.
Another problem with pathological assessment is an intraobserver variety. Except for skill
ful operator, an experienced pathologist is essential for proper evaluation of the samples.
However, differences in the assessment between to various pathologists are difficult to
avoid even with the use of validated scoring systems.
In recent years many non-invasive methods of liver assessment were developed. Imaging
techniques allow to describesteatosis, focal changes, malformations, inflammatory processes
of bile ducts and advanced fibrosis. Mild changes are, however, still difficult to detect. Elas
tography has been developed to evaluate liver stiffness being a useful tool to assess liver fib
rosis [9]. Fibrosis is also evaluated by different serological markers and panels of direct and
indirect markers or combination of both. Various cut-offs of the markers to detect advanced
fibrosis and cirrhosis were validated in numerous studies [4]. Nevertheless, problem with
intermediate stages of fibrosis still exist, since in such cases serological markers overlap.
Attempts to completely replace the biopsy with other non-invasive methods are not effec
tive as the collection of adequate liver sample and proper histological evaluation allows to
determine the extent ofthe liver damage and helps to establish the diagnosis.
5. Conclusions
Percutaneous liver biopsy is safe even in small children. Although severe complications are
rare, patients require frequent monitoring. Ultrasound guidance seem to reduce the number
of complications. Remaining a golden standard, the liver biopsy has certain limitations and
drawbacks that influence the results.
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Acknowledgements
This paper was supported by a grant from The Ministry of Science and Higher Education
No NN407 012036 to A. Mania
Abbreviations
AIH autoimmune hepatitis, CHB chronic hepatitis B,CHC chronic hepatitis C, NAFLD
non-alcoholic fatty liver disease, HUO hepatitis/hepatomegaly of unknown origin,
Author details
Anna Mania1, Pawe Kemnitz1, Magdalena Figlerowicz1, Aldona Woniak2 and
Wojciech Suewski1
1 Department of Infectious Diseases and Child Neurology, Faculty of Medicine, University
of Medical Sciences in Poznan, Poland
2 Chair of Clinical Pathology, Faculty of Medicine, University of Medical Sciences in Pozn
an, Poland
References
[1] Sporea I, Popescu A, Sirli R. Why, who and how should perform liver biopsy in
chronic liver diseases. World J Gastroenterol2008; 14(21): 3396-3402
[2] Cholongitas E, Senzolo M, Standish RA, Marelli L, Quaglia A, Patch D, Dhillon AP &
Burroughs AK. A systemic review of the quality of liver biopsy specimens. Am J
ClinPathol 2006; 125: 71021
[3] El-ShabrawiMH El-Karaksy HM, Okahsa SH, Kamal NM, El-Batran G, Badr KA.
Outpatient blind percutaneous liver biopsy in infants and children: is it safe? Saudi J
Gastroenterol. 2012;18:26-33.
[4] Mania A, Kemnitz P, Mazur-Melewska K, Figlerowicz M, Suewski W. Non-inva
sive assessment of liver serum markers and imaging techniques. In: Takanashi H.
(ed.)Liver biopsy. Rijeka: InTech; 2011. p265-282.
[5] Potter C, Hogan MJ, Henry-Kendjorsky K, Balint J, Barnard JA. Safety of pediatric
percutaneous liver biopsy performed by interventional radiologists.J PediatrGas
troenterolNutr. 2011;53:202-6.
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[6] Lebensztejn DM, Kaczmarski M, Sobaniec-Lotowska M, et al (2000). Blind liver biop

sy in childrendiagnostic significance and complications in authors own material.
Med SciMonit 6:11551158.
[7] Nobili V, Comparcola D, Sartonelli MR, Natali G, Monti F, Falappa P, Marcelini M.
Blind and ultrasound-guided percutaneous liver biopsy in children. PediatrRadiol
2003; 33:772-775.
[8] Kemnitz P, Mozer-LisewskaI,Mania A, Michalak M, Wozniak A, Kowala-Piaskowska
A, Figlerowicz M, Pohland J, Suewski W. Clinical importance of transcutaneous
needle biopsy of the liver in children with chronic hepatitis B. Exp. ClinHepatol 2009;
5;38-43.
[9] Castera L, Vergniol J, Foucher J, Le Bail B, Chanteloup E, Haaser M, Darriet M, Cou
zigou P & De Ledinghen V. Prospective comparison of transient elastography, Fi
brotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C.
Gastroenterology 2005; 128: 343-350.
83
Chapter 5
Risks and Benefits of Liver Biopsy in Focal Liver Disease

Letiia Adela Maria Streba,
Eugen Florin Georgescu and Costin Teodor Streba
http://dx.doi.org/10.5772/52620
1. Introduction
Even with the recent evolution of imaging techniques, and with the ever-increasing role of
serum markers, direct analysis of tissue samples maintains its role in modern medicine. This
is especially true for the diagnosis and assessment of the prognosis and evolution of a series
of viral, tumoral and inflammatory liver diseases. Thus, liver biopsy and histological assess
ment of the liver parenchyma can still be called by many the gold standard in diagnosis
and staging of associated disease. However, liver biopsy in itself implies a series of risks and
inherent discomfort for the patient. With the increasing availability of other non-invasive
methods routinely used in diagnosis and staging of liver-related diseases, many debate the
necessity and ethical implications of tissue sampling.
In the following pages, we will try and synthetize the historical evolution of liver biopsy,
describe the techniques used over the years and present its current recommendations and
their alternatives, with focus on the so-called virtual liver biopsy techniques currently
employed.
2. Historical landmarks and recent developments in liver biopsy

The first written documented report of a successful liver biopsy was made by Paul Ehr
lich in the book On diabetes published in 1884. He published an account of the proce
dure performed in 1880 in Berlin, along with graphical illustrations of the instruments
and the liver samples collected. This came detailed account was based on previous theo
retical advantages of this technique discussed by the French physician AGM Vernois in
1844, who in turn based his assumption on successful procedures performed for punctur
86
ing purulent echinococcus, as early as 1825 (Rcamier) and 1833 (Stanley). Cytology was
reported as a diagnosis method for liver disease by L. Lucatello (in Rome) in 1895, while
F. Schupfer performed liver and spleen biopsies with a thicker needle twelve years later,
in 1907. This new approach provided cylindrical-shaped tissue samples which could be
histologically prepared and analyzed [1].
Other scarce accounts of successful procedures followed in the next couple of decades (Oli
vet, 1926; Huard 1935; Silverman, 1938; Baron, 1939; Kofler, 1940; Dible, 1943), using differ
ent aspiration techniques performed with different modified biopsy needles [1].
A new stage in modern liver biopsy techniques was reached when, in 1957 and repeated in
the following year, Menghini performed and reported on the first one-second needle biop
sy performed with a special small caliber needle with no trocar and a sharp bevel. This was
the first time needle liver biopsy was introduced worldwide as a praised diagnostic techni
que capable of providing enough histological material for an accurate interpretation of the
pathological changes present in the parenchyma [1].
Following this radical advancement, liver biopsy became more spread and the technique
evolved once modern imagistic methods allowed for better and safer puncturing of the
liver parenchyma. Thus, the technique entered the image-guided age of investigation per
formed under computed tomography (CT) or ultrasound (US) real-time screening. Re
ports from Denmark, China, the United Kingdom, France or the United States of
America populated the 19601980 literature, once the technique became widespread and
fully acknowledged by the academic community. Its utility in diagnosing liver diseases
and later on in staging hepatitis or malignancies was undisputed for entire decades of
the 20th century [1].
Recent advancements, based on the advent of new imagistic high-accuracy techniques based
on both US and CT/RM approaches, highly diminished the role played by this invasive in
vestigation. The term virtual biopsy became more and more present in recent literature,
once both doctors and patients alike became more confident and were introduced to these
high-yield methods, such as Transient or Acoustic Radiation Force Elastography. Moreover,
advanced serum markers (such as, for example, the Fibrotest-Actitest battery of tests) allow
for an accurate non-invasive staging in hepatitis. The introduction of arterial uptake con
trast-enhanced US and CT/RM techniques substantially decreased the role of biopsy in diag
nosing liver biopsy [24].
However, histology remains one of the most accurate methods for evaluating liver paren
chymal changes, and is always used in malignancies when the diagnosis is uncertain or
when other non-invasive methods fail to provide an accurate staging for hepatitis. Along
with these non-invasive techniques came a revolution in in-situ biopsy methods. Such is
probe-based confocal laser endoscopy (pCLE), which uses miniaturized probes connected to
a laser source through fiber optics, small enough to fit inside a biopsy needle, thus provid
ing rapid live assessment of liver architecture [5].

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3. Modern liver biopsy techniques and sampling adequacy

3.1. Percutaneous biopsy
All modern percutaneous liver biopsy techniques have rapidity as a common denominator.
Either cutting or suction needles can be used for transthoracic or subcostal biopsy, either af
ter palpation or imaging assessment of the puncturing zone, or, preferably, under continu
ous image guidance. The transthoracic approach is the preferred method used, under realtime US or (more rarely) CT guidance and after a thorough imaging investigation of the
liver and puncture route. All percutaneous methods imply two phases, one extra-hepatic
corresponding to the needle puncturing the skin and reaching the needle, and a hepatic
stage in which the needle passes the liver capsule, collects the parenchyma material, and is
swiftly extracted. It is considered a relatively safe procedure, complication rates varying be
tween studies, from 0.75% up to 13.6% [6].
Trucut needles and their modified versions driven by spring-loaded biopsy guns are in
creasingly used and are the instruments of choice in many centers worldwide, especially in
Europe [7]. Needle diameters vary between 1.20 mm to 1.60 mm, smaller calibers being used
when a high risk of complications is suspected.
Suction needles are less expensive and their operation allows for rapid intra-hepatic han
dling, thus being easier to use and possibly imply less bleeding-related complications. The
most widespread types are the Menghini, Jamshidi and Klatskin needles, which remained
virtually unchanged since their introduction in the second half of the last century. The maxi
mum required time for a complete syringe suction of the cytological material and the con
secutive needle retraction is 0.5 seconds. The intrahepatic phase is reduced to as low as 0.1
seconds when the needle is operated by an expert practitioner [8].
Image guidance has become mandatory in centers where the gastroenterologist can perform
his or her own US exam. Real-time surveillance of the procedure greatly decreases the risk
of complications (such as bleeding) and minimizes post-procedural complaints such as pain
or hypotension. Hepatologists in the United States usually prefer to have a radiologist per
forming the procedure under CT or US guidance [8].
3.2. Transjugular (transvenous) biopsy
The transjugular route is preferred when the risk for complications is high and therefore a
percutaneous approach is not considered safe enough for the patient. Patients with clinical
ascites, known hemostatic defect, cirrhotic liver with clinical signs of organ deficiency
(smaller size and increased palpatory stiffness) or morbid obesity are usually prime candi
dates for this approach. Another situation when the transvenous approach is preferred is
when additional pressure measurements in the hepatic vein are required [8].
The resources needed for this procedure are higher than percutaneous approaches; howev
er, complication rates are lower (2.5% up to 6.5%) according to some authors [9], with mor
tality rates of approximately 0.09% in high-risk patient groups [10]. The expertise of the
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88
performing physician also plays a crucial role in the success rate of this procedure, and
should be considered along with the higher resource costs when choosing this access route
for a lower-risk patient [1].
Another very important aspect is the lower quality of the tissue specimens collected through
the transjugular approach. The tissue cylinders are thinner and more fragmented than those
obtained through percutaneous biopsy, and usually represent only 1-2 cm of the liver paren
chyma, containing fewer portal fields [11].
3.3. Surgical or laparoscopic biopsy: Novel approaches for liver biopsy
This approach is preferred in patients with peritoneal involvement when an abdominal
cancer is present, with associated ascites or peritoneal disease with ascites of suspected
hepatic origin. Also, focal hepatic lesions can be targeted for biopsy through the laparo
scopic channel.
Biopsy can thus be performed with either normal needle systems, or by wedge resection.
However, the later approach may overestimate the level of fibrosis, as the resection is per
formed too close to the fibrotic capsule that envelops the liver. The procedure is always con
ducted under general anesthesia and requires controlled pneumoperitoneum by infusion of
nitrous oxide, always performed by trained physicians, allowing for a good control of bleed
ing and a minimum set of complications due to the large working area created. In direct
comparison with percutaneous biopsy, the laparoscopic approach provides a higher level of
accuracy as it allows the evaluation of the surrounding peritoneum [12]. The main complica
tions are related to the general anesthesia used for the procedure, the local abdominal and
intra-peritoneal traumas associated, as well as the risk of bleeding, which is also present in
the other types of biopsy.
Advancements to surgical techniques led to the development of the natural orifice translu
minal endoscopic surgery (NOTES), a new surgically-derived endoscopic technique that
uses a transgastric or transanal route to facilitate the access to the abdominal cavity. One re
cent study presented a liver biopsy performed through a transgastric flexible endoscopic de
vice which permitted the inspection of the liver and surrounding intraperitoneal space. The
technique can be applied to morbidly obese patients or to patients at high risk of complica
tions [13]. This approach remains however limited at the present time to a few highly select
ed patients, and is performed only by trained surgeons and gastroenterologists, at moderate
to high costs and in selected centers.
Recent studies also focused on evaluating the liver capsule in cirrhotic patients through
pCLE inserted through a laparoscopic channel, this being a promising field in the advance
ment of minimally invasive biopsy techniques [14]. Another study describes the use of pCLE
in a routine minilaparoscopy setting, performed under conscious sedation. The authors
could describe subsurface serial images in real time, allowing for an in vivo analysis of the
liver parenchyma [5]. This approach may lead the way to targeted biopsy through live as
sessment of the liver parenchyma, as well as immediate morphological and dynamic evalua
tion of intrahepatic structures.

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3.4. Adequacy of liver biopsy samples

Analysis of the biopsy material under ultraviolet fluorescent light may be required in order
to identify porphyria. Liver tissue obtained through biopsy is then quickly transferred into a
buffer solution, usually 4% or 10% neutral formalin, to avoid the alterations it may sustain
due to hepatic enzymes autolysis. It can then be subjected to various preparation techniques,
in accordance to what diagnostic tests will follow with that specific sample (frozen section,
RNA detection etc.) [1].
An adequate biopsy fragment is between 1 and 4 cm long, weighting between 10 to 50 mg,
with a minimal diameter of 1 mm. Fragmented samples from Menghini needles are accepta
ble, as their added size is somewhere in the vicinity of 2 cm (usually range from 1 to 2.5 cm
in length). In order to properly represent the parenchymal architecture, at least 1011 portal
tracts should be completely present, six being a minimally acceptable number. Specimens of
inadequate lengths usually lead to understaging of fibrosis and underestimate the grade of
inflammation. Cirrhotic parenchyma usually comes fragmented through biopsy, thus lead
ing to approximately 20% sampling errors [15, 16].
As it is appreciated that a liver biopsy specimen represents 1/50 000 of the total organ mass,
discussions regarding how representative it can be for diffuse lesions always existed in the
literature [8, 17]. It is however appreciated that most diffuse (steatosis or inflammation etc.)
or focal lesions (both malignant and benign), as well as structural lesions such as fibrosis can
be visualized with a fairly high degree of accuracy, if the minimum amount of liver paren
chyma and the required number of portal spaces are present. It was however demonstrated
that the size of the sample is directly correlated to an underestimation of inflammatory
changes [18], this paradigm being extended to fibrotic changes and has a direct effect on the
subsequent grading and staging [1, 19, 20].
Another issue highly debated in literature is the inter-observer variability; even with the
wide usage of quantification scores for both inflammation and fibrosis such as the Knodell
[21] scoring system and the revised Ishak version [22] or the METAVIR score [23]. All inter
pretations are subjected to the experience and training of the pathologist, which is an inde
pendent variable in itself, separated from the inherent sampling and procedural errors. A
second opinion is always recommended, and two pathologists are usually present in most
large referral centers. Collaboration between the pathologist and the clinician performing
the liver biopsy is also preferred, as some studies indicated [2426].
The most important quantification parameters refer to its geometry and relationship be
tween the principal compartments portal tracts and the elements of the arterial vascular
system; the configuration adopted by hepatocyte plates; the sinusoids and the perisinusoi
dal compartment; the amount of connective tissue, fat and the number of ducts present, as
well as other normal cellular infiltrates of lymphoid origin [8]. Regenerative nodular hyper
plasia or macronodular cirrhosis can be sometimes classified as normal parenchyma, and
the inherent variations of normal inflammatory cellular infiltrate can be misleading for an
inexperienced pathologist when observing low grade inflammatory lesions [8, 27].
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4. Risks, complications and post-procedural complaints of liver biopsies

The main risks for a patient subjected to liver biopsy were already briefly discussed in the
previous paragraphs. Their frequency and predisposition in certain patient groups are de
terminant factors for choosing one biopsy technique in favor of another. The risk of bleeding
cannot be excluded with any instrument, and liver biopsy is not recommended in most cases
of suspected primary liver cancers because of a needle track seeding of tumor cells. These
however do not exclude liver biopsy as a last resort diagnostic tool, when imagistic or serum
tests proved constantly inconclusive or do not converge to an outcome.
The most commonly occurring complication of percutaneous liver biopsy is pain, present in
up to 84% of procedures and ranging from mild discomfort to severe pain [28]. It is usually
located in the right upper quadrant and it is referred to the right shoulder, with various in
tensities and time of installment. Moderate to severe pain is present in fewer than 5% of all
patients, and may be the sign of a more severe complication such as bleeding or the punc
turing of the gallbladder [16, 29]. Mechanisms that lead to pain after the biopsy maneuver
are not fully understood, however it is likely to be caused by bile or blood extravasation
with subsequent capsule swelling (the only liver component with sensitive nervous termina
tions) [30]. Another cause of upper abdominal pain is the traction of the falciform ligament
after the puncture. Cervical pain, as well as pain in the right shoulder, may also be caused
by the irritation of the phrenic nerve. Subcapsular hematoma may lead to respiratory pain
and irritation of the pleura or peritoneum may lead to vagal stimulation and consecutive va
gal shock, manifested through bradycardia, severe hypotension, weak pulse and intense
pain in the upper abdomen [1]. In some cases of extreme pain, hospitalization and further
imaging tests are required to determine the correct course of action for these patients.
However, the most important complication of liver biopsy is bleeding. The most severe
bleedings occur intraperitoneally, when they determine a drop in vital signs and can be vi
sualized through imaging [16, 31]. Urgent hospitalization and blood transfusion, even fol
lowed by surgery or radiological intervention may be required. Nevertheless, these cases are
scarce, with 1 in 2 500 up to 10 000 biopsies incidence, while less severe cases which do not
require blood transfusions or surgical maneuvers are more frequent, approximately 1 in 500
biopsies [16]. Serious bleeding-related complications usually occur within 2 hours of the
procedure, and over 90% of all bleedings become evident within 24 hours of the procedure.
Clinical symptoms are revelatory, as patients experience hypotension and shock. Age and
the underlying conditions also are predictive factors, as older patients and liver masses are
more frequently associated with post-puncture bleeding. A correlation between the needle
type and the risk for bleeding was also cited in literature, as cutting needle seem to pose an
increased risk compared to their suction counterparts [15]. Other factors are related to oper
ator experience, the diameter of the needles and their diameter [16].
A correlation between conventional coagulation tests and the risk of bleeding has not been
sufficiently demonstrated until now; therefore no certain recommendations in this regard
are currently in place [16]. The option to insert coagulation agents on the needle tract is con
sidered, especially in the US, with no definite data on its ability to prevent possible bleed

http://dx.doi.org/10.5772/52620
ings. As already mentioned, the transvenous approach is preferred in certain categories of

patients as it is considered safer, even though several pooled analyses showed similar risks
with standard percutaneous methods [10,16].
The singular major complication of liver biopsy, caused in turn by consecutive severe bleed
ing is patient death. No consistent data regarding post-procedural mortality exists in the lit
erature, the most commonly quoted rate being less or equal to 1 in 10 000 biopsies [16], and
seems to be greater after biopsies of malignant liver masses compared to diffuse parenchy
mal disease [6].
Other complications of liver biopsies include the perforation of other viscous organs, bile
peritonitis (major complication which can result in death), infections (especially in posttransplant patients due to immunosuppressive medication), hemobilia, pneumothorax (in
stantly recognized on radiographs, essentially to diagnose quickly due to high risk of death)
or hemothorax. Correct usage of imaging methods both when choosing the biopsy site and
for surveillance of the procedure minimizes many of these risks, especially those related to
puncturing adjacent structures [16]. The risk of needle track seeding when puncturing liver
malignancies exists in 1 to 3% of all cases [32], as will be detailed below.
5. Current recommendations regarding conditions that require liver

biopsy
The indications for liver biopsy were greatly reduced since the recent introduction of accu
rate non-invasive tests which can evaluate liver parenchyma with minimal or no patient
trauma. The concept of liver biopsy may evolve even further, if in vivo direct histological
methods such as pCLE will provide important additional data. It is most likely that the rec
ommendations for liver biopsy will suffer further changes in following years. A series of
these advancements will be discussed separately within this chapter. Below, we will de
scribe some of the main indications for liver biopsy, either for diagnostic purposes or for
evaluating and staging liver disease.
5.1. Grading and staging of chronic viral hepatitis
The recent outburst of viral hepatitis cases (especially as a result of the increasing number of
newly diagnosed virus C infections) represents a major health burden worldwide. With al
most four million people being infected in the United States alone, and between 130 and 170
million worldwide, chronic hepatitis C virus (HCV) infections and more than double those
figures for hepatitis B virus (HBV) infections, this ensemble of viral diseases currently repre
sent the main cause of liver-related morbidity [33, 34].
Nowadays, the role of liver histology in the positive diagnosis of chronic viral hepatitis has
greatly diminished. However, it still plays a central role when assessing both activity and
progression of the disease [8, 35]. Sampling issues arise when evaluating liver parenchyma
affected by chronic hepatitis, as the quality of the obtained specimens can greatly influence
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the semi-quantitative scores developed in the last four decades to quantify disease progres
sion. There are a number of changes present within the liver and their heterogeneity makes
the 10-complete portal spaces paradigm essential when evaluating disease severity. All
scoring systems are bound to yield significantly different results, primarily because of sam
ple variability, but also as a result of the different levels of expertise from the pathologist
involved in their evaluation. All modifications of the liver parenchyma inflammation, ne
crosis or fibrosis exhibit particularities and can be subjectively interpreted even in a scor
ing system [8].
The first approach to liver biopsy scoring for chronic hepatitis dates from the early 1980s
when the histological activity index (HAI) was introduced by Knodell and Ishak [21]. This
model did not clearly delimited between disease grades (that is, the importance of any in
flammatory activity present) and stage, which refers to the degree of fibrosis and parenchy
mal remodeling. The later modification performed by Ishak resolves most of these issues
and is currently used worldwide, partially replacing or at least complementing the earlier
alternative Knodell classification. The preferred approach is a parallel evaluation using sev
eral scoring methods, such as the modified HAI, the Scheuer or the Ludwig systems and the
Knodell classification, or the METAVIR algorithm devised in France [23].
5.2. Abnormal hepatic biochemical tests, alcoholic and non-alcoholic liver disease
Chronically elevated hepatic biochemical parameters are a common concern for many pa
tients during routine screenings or general consults. Gastroenterologists facing abnormal as
partate aminotransferase/alanine aminotransferase, gamma-glutamyltransferase or alkaline
phosphatase levels have to conduct a thorough anamnesis to determine the underlying con
dition. Many such patients either acknowledge high alcohol consumption or are diagnosed
with non-alcoholic liver disease (NAFLD) associated with their lifestyle, while few remain
undiagnosed until they begin to display signs of liver cirrhosis (cryptogenic cirrhosis or cir
rhosis of unknown etiology). The latter two classes are usually diagnosed through liver bi
opsy, as no other condition can be found from either their background or non-invasive
investigations and blood tests [8, 16].
The most common aspect revealed by liver biopsy in these patients is macrovesicularsteato
sis, intracellular lipid accumulation exceeding 5% of the total cellular population. This mac
rosteatosis is generally coined as fatty liver disease (FLD) and can either be identified as
either alcoholic liver disease (ALD), when regular alcohol consumption above established
thresholds is established, or NAFLD when obesity, type 2 diabetes mellitus and/or hyperli
pidemia are associated. Steatohepatitis, either of alcoholic origin (alcoholic steatohepatitis
ASH) or metabolic (non-alcoholic steatohepatitis NASH) share histological similarities.
NASH is recognized as a form of NAFLD with ballooning hepatocytes and necroinflamma
tory changes, as well as fibrosis and parenchymal remodeling. The NAFLD activity score
(NAS) was developed in an attempt to objectively quantify the extension of this disease.
This score sums the three pathologic features steatosis, lobular inflammation and hepato
cellular ballooning on a 0 to 8 scale, 5 being the cut-off point for a certain diagnose of NASH
and 34 being labeled as borderline steatohepatitis [36, 37].

http://dx.doi.org/10.5772/52620
Currently, even though liver biopsy is still regarded as the gold standard when diagnos
ing these conditions, no consensus has been reached. Liver biopsy remains therefore a con
troversial decision which ultimately has to be performed only when a clear diagnosis cannot
be extracted from serum values, imagistic findings and clinical features [38].
5.3. Metabolic liver disease
Diseases that determine intrahepatic iron accumulation are the main indications for liver bi
opsy when a metabolic condition is suspected, besides NAFLD or ALD. Hereditary hemo
chromatosis, in its various forms identified today, is routinely diagnosed and staged
through liver biopsy [8, 39]. The metabolic syndrome (syndrome X) represents the increased
accumulation of iron within hepatocytes, in the context of NAFLD. These deposits are not
distributed equally among various regions of the liver, therefore deeper biopsies are needed
in order to collect more tissue for analysis [8, 40]. For this purpose, at least two scores are
currently used the Deugnier and the Brissot scores [41, 42]. The hepatic iron index is calcu
lated through a mathematical formula which takes into account the hepatic iron concentra
tion (evaluated by liver biopsy), its atomic weight as well as the age of the patients. An
index above 1.9 is an indicator of hemochromatosis; however its sensitivity is low as it is de
pendent on the timing of the liver biopsy [8].
5.4. Focal liver lesions
Discovery of a focal liver lesions (FLL) can occur after imaging tests used routinely for either
screening or diagnosis. The practitioner may encounter lesions of various sizes, number and
location, some of them being associated with pre-existing conditions. This is especially the
case of primary liver malignant tumors, either hepatocellular carcinoma (HCC) or cholan
gyocarcinoma (CC). Early discovery of a FLL is possible in up to 60% of all cases, especially
in developed countries where surveillance programs are well established and health serv
ices are available to the majority of the population, irrespective of their location and eco
nomic status [43, 44].
Imaging alone is currently the main diagnostic procedure for HCC, as modern contrast-en
hanced techniques, either by CT or MRI, are sufficient to highlight the hallmark pattern of
tumor vascularization. Diagnostic criteria in the United States of America, Europe and Asia
stipulate that imaging techniques are sufficient to diagnose the majority of HCC lesions, bi
opsy being reserved for the few situations where imaging is unclear, discordance between
two methods exists, or tumor size does not allow a precise imaging diagnosis [4345]. A de
fining criteria for evaluating FLLs is the presence of an underlying hepatic condition such as
hepatitis or cirrhosis.
When HCC is suspected in cirrhotic patients, criteria for liver biopsy are set by the size of
the tumor. In nodules between 1 and 2 centimeters, diagnosis should ideally be based on
non-invasive criteria; however, confirmation through biopsy should be sought whenever
possible. The evaluation should be performed ideally by a pathologist with extensive experi
ence in evaluating liver biopsies. In case of inconclusive findings after the initial biopsy, a
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second one should be performed if no other imaging criteria are present during the evalua
tion period. Nodules larger than 2 centimeters discovered through routine US should be ide
ally diagnosed through non-invasive procedures; however, when radiological findings are
atypical, a liver biopsy should be obtained as confirmation [4345]. A panel of immunohisto
chemical markers was proposed as diagnostic when evaluating liver biopsies for HCC. A
combination of glypican 3, heat shock protein 70 and glutamine synthetase are recommend
ed for the differential diagnosis between early HCC and high grade dysplastic nodules [46]
(Di Tomaso et al, 2009). A final recommendation of the EASL-EORTC guidelines is that liver
biopsy should be performed within controlled settings of scientific research, for identifying
new markers for HCC and for tissue bio-banking[44].
The current tendency in diagnostic medicine is to avoid liver biopsy when evaluating HCC
[44]. The main reasons against performing liver biopsy are the high rate of sampling errors
which would diminish the sensitivity of the investigation; a higher rate of recurrence posttransplant in patients who underwent liver biopsy and finally the small but well-established
risk of needle track seeding. In transplant referral centers, liver biopsy is performed more
frequently, as there is an increased need for a correct final diagnosis; however, these proce
dures are subject to wide variation depending on country-specific regulations [43, 44]. An
other argument for liver biopsy in HCC cases that benefit from chemotherapy would be the
importance of histological grading. Response to local or systemic anti-angiogenic or antiproliferative agents might be dictated by the microscopic configuration of the tumor and the
amount of angiogenesis markers present on histological samples [16].
The second most important primary liver malignancy is CC. It can also develop in the pres
ence of an underlying liver condition, such as chronic biliary tract diseases. Imaging diagno
sis is sometimes difficult, as it may present similar contrast-enhancing patterns to those of
HCC the majority of CCs are solitary masses present in the hilum, while a minority can
develop in other regions [43, 44]. Mixed forms of CC/HCC may also be present, their noninvasive diagnosis being even more difficult. All these forms of either atypical CCs or mixed
presentations are usually subjected (with various degrees of variability, depending on set
ting and context) to liver biopsy. Surgical intervention, either by resection or liver trans
plant, are the approaches that yield the best survival chances for the patient. Therefore, liver
biopsy may be indicated, as well as concomitant biopsy of lymph nodes in the upper ab
dominal area [16].
Metastases have the overall highest incidence amongst malignant liver lesions [47]. When a
secondary malignant liver lesion is suspected and the physician cannot identify the primary
point, liver biopsy is usually diagnostic, even when imaging fails to provide enough detail.
If an underlying parenchymal disease is also suspected, biopsy should be performed outside
the lesion site as well, for an extended and more precise diagnosis. A vast panel of markers
may be employed in an immunohistochemistry study; however, the histologic architecture
identified through normal techniques may be sufficient for an expert pathologist to deter
mine the primary site of origin [1, 16].
Other rare primary liver parenchyma or bile duct malignant or benign neoplasms can ulti
mately be identified through histological analysis, after careful imaging-guided liver biop

http://dx.doi.org/10.5772/52620
sy is performed. This diagnosis is often not possible on cross-sectional imaging studies as

well as tumor serum markers, as their specificity for such lesions is inadequate. An expert
hepatologist should closely collaborate with an experimented pathologist, as the diagnosis
is difficult most of the times. These lesions may develop in the presence of an underlying
liver condition, which would aid the clinical diagnosis or suspicion on the part of the
clinician [1, 16].
The majority of lesions discovered through imaging techniques in patients without pre-ex
isting liver conditions are benign in origin, mostly solitary or occasionally multiple. They ex
hibit particular vascular patterns in contrast-enhanced imaging techniques and are thus
easily diagnosed without the use of invasive techniques. Such is the case of liver hemangio
mas, mostly solitary benign tumors with characteristic contrast enhancement throughout all
phases of an imaging investigation. Other lesions such as focal nodular hyperplasia are also
usually solitary and may display distinct features such as central scarring or particular en
hancement patterns (spiked wheel enhancement etc.). All these particularities have a mor
phological substrate: central hypoechoic areas which do not show vascular
hyperenhancement usually correspond to areas of necrosis; intense signal enhancement
zones are indicators of high microvessel density and neo-angiogenesis vessels; the US or CT
peripheral rim translate in certain particularities of fibrous capsules [1, 16, 44].
Overall, lesions may present as cystic, solid or vascular; all these particularities usually be
ing identified through non-invasive procedures prior to liver biopsy. In the USA for in
stance, liver biopsy is performed by imagists as they can perform the pre-biopsy or real-time
assessment of the procedure, while in Europe most gastroenterologists or hepatologists per
form the procedure themselves, under US surveillance [43, 44]. A core biopsy is usually pre
ferred to fine-needle aspiration, as histology is considered superior from a diagnostic
perspective compared to cytology; another reason being that experts in evaluating histology
are more numerous compared to cytologists. The risk of puncturing blood vessels, either
major arteries in the normal parenchyma, or intra-tumoral vessels is considerably diminish
ed by real-time imaging guidance, for instance US with color Doppler. The risk of track
seeding exists, even if extremely low (one study estimates a risk of 0.13%, while in other
studies no such incidents were reported) [48, 49]. A certain dependency on the technique
and size of the needle was also proven [50]. Infectious lesions may be biopsied; even if echi
nococcal cysts were considered an absolute contraindication as puncturing can be associated
with anaphylactic shock and death, it was proven that these lesions can be aspirated with 19
or 22-gauge needles, taking all preparations for possible anaphylaxis [51].
6. Novel techniques in liver biopsy; modern non-invasive alternatives

6.1. Probe-based confocal laser endomicroscopy
The latest development in histological evaluation of gastrointestinal structures is confocal la
ser endomicroscopy. It allows for the in vivo evaluation of dysplasia and malignancies of
the gastrointestinal tract, or in order to obtain directed biopsies that would allow rapid and
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more precise diagnoses [52, 53]. The first embodiments of this technique required dedicated
endoscopes to be used for evaluating cavitary structures accessible from both ends of the di
gestive tracts.
Recent advancements however were able to miniaturize the technology so the imaging mi
croprobe can be connected to 30,000 fiber-optic threads that enable point-to-point real-time
detection at 12 frames/sec. The imaging device by itself measures less than 1.5 millimeters in
diameter, thus allowing its use through 19G or tru-cut biopsy needles, or insertion by lapa
roscopy or NOTES [53]. This technology will allow in vivo, real-time imaging of liver histol
ogy, technically enhancing the capabilities of liver biopsy [54]. A few studies on animal
models exist in the literature, detailing pCLE use for liver histological imaging [14, 55, 56].
The technique can be used for assessing the state of hepatocytes and the morphology of the
liver tissue, or can be limited to the study of the exterior liver capsule, yielding interesting
preliminary results in the setting of cirrhosis. Mennone et al reported interesting results re
garding a fibrotic pattern and collagen deposits in animal models with cirrhosis induced by
bile duct ligation [14]. The technology shows promise and may someday allow for safer his
tological assessment of patients with chronic liver disease irrespective of its advancement,
either cirrhotic or having any extreme complications, such as HCC.
6.2. Non-invasive imaging and serum tests for the assessment of fibrosis
Transient elastography (TE, Fibroscan developed by Echosens, Paris, France) and Acoustic
radiation force impulse (ARFI) are two ultrasound-based methods for quantifying liver fib
rosis without the need for histological assessment. Another approach is through serum
markers of fibrosis quantification, processed in complex mathematical formulas which give
a quantitative result for liver stiffness, such as the Fibrotest, Biopredictive and the aspartate
transaminase to platelets ratio index (ARPI) approaches.
TE is a novel and rapid non-invasive examination which involves minimal patient discom
fort over a relatively low time period (one examination may take up to 5-10 minutes de
pending on the skeletal and adipose conformations of the patient). The device consists of a
hand-held vibrating unit with an ultrasound transducer probe mounted on its axis, which
generates medium amplitude vibrations at a low frequency, thus inducing an elastic shear
wave in the underlying tissue. The hand-held probe is connected to a modified tower US
machine which registers the result and through the on-screen software interface presents the
user with an elastogram as a function of depth in time. The patient lies on his/her side and
the probe is placed against the skin on the median clavicle line, directed towards the ana
tomical location of the liver, at a 90 degrees angle with the skin surface. Its results are pre
sented as kilo Pascals (kPa), units of applied force. A series of 10 measurements are
mediated to present a final value of the liver stiffness, which is equivalent to an F-stage fib
rosis measurement obtained through biopsy [2].
ARFI is another technology that uses short-duration, high-intensity acoustic pulses which in
turn exert mechanical excitation upon the tissues, generating local displacement resulting in
shear waves. Their velocity can be assessed in a selected cylindrical area of interest of 0.5 cm

http://dx.doi.org/10.5772/52620
(length) x 0.4 cm (diameter), up to 5.5 cm below skin level. Its results are expressed as veloci
ties, in m/s [4].
Fibrotest-Actitest (Biopredictive, France) is a serologic marker-based algorithm which repre
sents an alternative to invasive biopsy techniques. It received clinical validation in patients
with chronic hepatitis B and C, ALD and NAFLD. Fibrotest consists of a panel of markers
designed for appreciating liver fibrosis: Gamma-glutamyltranspeptidase (GGT), Total biliru
bin Alpha-2-macroglobulin, Haptoglobin, and Apolipoprotein A1. Necroinflammatory ac
tivity is appreciated through the Actitest component, which adds Alanine transaminase
(ALT) to the above mentioned serum markers [3, 57]. All these tests are performed in vali
dated laboratories due to their complexity and variability of their different components and
their results are inserted in a complex mathematical formula through a web-based interface,
the end-result being correlated with other quantitative score systems such as METAVIR,
Knodell or Ishak [58].
The best results are provided by a combination of two or more non-invasive methods, one
study in particular finding that Fibrotest and Fibroscan offers the best diagnosis perform
ance compared to liber biopsy as a gold standard, at least for advanced fibrosis (F values
beyond 2) or cirrhosis (F3 or F4) [2]. This conclusion was reached by another, more recent
study performed by Boursier and his collaborators [59]. They diminish the number of pa
tients who require liver biopsy, however, this procedure is not excluded in all cases. Some
studies have shown a high variability between Fibroscan results, dependent of the bodymass index and population factors [60, 61]. A discordance between liver biopsy staging and
the estimation provided by non-invasive methods has also been identified [34]. It was ap
proximated that 3040% of all patients investigated by a combination of non-invasive imag
istic and marker-based methods still require liver biopsy, during either sequential or
simultaneous protocols [60, 61].
7. Conclusion
Despite all its limitations and the advances in modern lesser invasive techniques, liver biop
sy remains the gold standard for evaluating a wide array of liver diseases.
The main concern when turning to tissue sampling through biopsy is the risk/benefit ra
tio, the decision ultimately belonging to the clinician involved. The risks may at times be
higher than the implied diagnostic outcome, in which case other methods are preferred
for the diagnosis.
Currently, it is recommended that all interpretations should be based on proper tissue
blocks, with the correct technique applied. It is preferred that more than one pathologist
with extensive experience in liver pathology should formulate the final histological diagno
sis. This is especially true for FLLs and liver malignancies, as benign features may at times
overlap, making the diagnosis uncertain.
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Modern imagistic techniques allow for precise non-invasive evaluation of liver fibrosis in
the context of hepatitis; however, the correct methodology for interpreting these tests is yet
to be established. Novel imagistic approaches may in time open new perspectives for liver
biopsy, by providing in vivo, real time data on liver parenchymal features which would
prove useful for accurate diagnosing of otherwise difficult to interpret pathologies.
Author details
Letiia Adela Maria Streba, Eugen Florin Georgescu and Costin Teodor Streba
University of Medicine and Pharmacy of Craiova, Romania
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ue of biomarkers (FibroTest) in patients with chronic hepatitis C. Clin Chem. 2006;
52: 188796.
[58] Imbert-Bismut F, Messous D, Thibault V, et al. Intra-laboratory analytical variability
of biochemical markers of fibrosis (Fibrotest) and activity (Actitest) and references
ranges in healthy blood donors. ClinChem Lab Med. 2004;42: 323333.
[59] Boursier J, Vergniol J, Sawadogo A, et al. Thecombination of a blood test and Fibro
Scan improves the non-invasivediagnosis of liver fibrosis. Liver Int 2009;29:1507
1515.
[60] Kim SU, Choi GH, Han WK, et al.What are true normal liver stiffness values using
FibroScan?: a prospectivestudy in healthy living liver and kidney donors in South
Korea.Liver Int 2010;30:268-274.
[61] Das K, Sarkar R, Ahmed SM, Mridha AR et al.Normal liver stiffness measure
(LSM) values are higher in both leanand obese individuals: a population-based study
from a developing country.Hepatology. 2012;55:584-593.
Chapter 6
Current Trends in Liver Biopsy

Indications for Chronic Liver Diseases
Jean-Franois Cadranel and
Jean-Baptiste Nousbaum
http://dx.doi.org/10.5772/52618
1. Introduction
At the present time, pathological examination of a liver fragment obtained by liver biopsy
remains an essential diagnostic tool of numerous chronic liver diseases [1] Indications for
liver biopsy (LB) have changed considerably over recent years due to the development of
sensitive and specific tests for diagnosis of several chronic liver diseases (i.e., serology for
hepatitis C, antimitochondrial M2 in primary biliary cirrhosis, genetic screening for he
reditary hemochromatosis), but also because of intensive development during the last dec
ade of non-invasive assessment of fibrosis using serum tests (FibroTest, FibroMeter,
APRI score) and/or by physical methods such as pulsed elastography (FibroScan), in
particular, for chronic hepatitis C. Ultrasound-guided liver biopsy is often necessary to ob
tain a tumor fragment in cases of suspected primary or secondary liver malignancy and
will not be discussed here [1]. In the present article, we will limit ourselves to indications
of liver biopsy in diffuse parenchymal disease of the liver and its relative and absolute
contraindications. Modalities for performing liver biopsy and complications will not be
discussed here. Liver biopsy is an invasive procedure with possible complications; thus,
individual benefits for the patient must be weighed against possible risks. Liver biopsy is
indicated when the expected amount of information obtained exceeds the risks related to
the procedure, when the diagnosis required for establishing a prognosis cannot be ob
tained without pathological examination of the liver, and finally, when the treatment deci
sion depends on pathological results [1].
104
2. Indications for LB
Indications for liver biopsy in chronic liver disease have evolved (Tables 1,2). The main ad
vantages of LB with respect to the etiology of liver disease are shown in Table 2 and will be
detailed later. The indication for liver biopsy is appropriate when the treatment or prognosis
will be modified by results of histopathological examination of the liver. However, liver bi
opsy is not appropriate when the therapeutic decision and/or establishment of a diagnosis
does not depend on histological findings [1].
Indications
For diagnostic purposes
- Combination of several parenchymal liver diseases - Abnormal liver tests of unknown origin *.
- Fever of unknown origin.
- Focal or diffuse abnormalities on imaging studies.
For prognostic purposes
- Assessment of known parenchymal liver diseases.
For research purposes
- Development of treatments related to results of histological analysis
* After complete check-up, according to (2)

Table 1. Indications for liver biopsy
Cause of liver disease
Diagnosis
Evaluation of
Prognosis
Management
fibrosis
Hepatitis B
+++
+ (+)
+++
Hepatitis C
+++
+ (+)
++++
(Non-invasive markers
of fibrosis)
Hemochromatosis
+/-
+++
+ (+)
Wilson's disease
++
+++
-1 Antitrypsin deficiency
+++
Depends on lung status +
+++
+++
++++
++
+++
+++
++
Primary sclerosing cholangitis
++
+/0
Alcoholic liver disease
+/-
++
++
Severe acute alcoholic hepatitis
+++
NA
NA
Steatosis / steatohepatitis
+++
+++
Infiltrative lesions of the liver
++++
NA
NA
Medicinal cause
++
NA
NA
+++
In particular,
seronegative
Primary biliary cirrhosis / overlap
syndrome
Follow-up after liver transplantation ++++
NA: not applicable

Table 2. Utility of liver biopsy in clinical practice for diffuse parenchymal damage
Current Trends in Liver Biopsy Indications for Chronic Liver Diseases

http://dx.doi.org/10.5772/52618
Etiology
1997 Study [31]
2009 Study [13]
of 2,084 biopsies
of 8,580 biopsies
(%)
(%)
Hepatitis C
54.1
33.6
Delta hepatitis B
5.8
14.4
Hereditary hemochromatosis
4.3
Cholestatic disease of the
4.3
3.7
3.5
Liver transplantation
12.
Miscellaneous
17.6
12.2
Metabolic steatopathy
unlisted
8.9
liver (primary biliary cirrhosis. primary

sclerosing cholangitis. chronic
cholestasis)
Table 3. Indications for liver biopsy. Evolutionary trend in France.
2.1. Chronic hepatitis C

Liver biopsy has long been the only reference for assessing necroinflammatory lesions and
fibrosis in hepatitis C. Liver biopsy is most useful for evaluating the existence of co-morbidi
ties: alcoholic liver disease, non alcoholic fatty liver disease and iron overload, which are es
pecially common in patients with chronic hepatitis C. In 2002, the French consensus
conference no longer recommended systematic liver biopsy in patients with consistently
normal transaminases [2]. At that time, for patients recently contaminated with genotype 2
or 3 infection, without co-morbidity and/or when the indication was viral eradication inde
pendently of fibrosis data, then antiviral treatment could be undertaken without requiring
LB [2]. Furthermore, liver biopsy is not useful when diagnosis of cirrhosis is obvious [2].
While abdominal ultrasonography is satisfactory for assessing the existence of steatosis, liv
er biopsy is needed in order to evaluate the existence of steatohepatitis, iron overload or al
coholic liver disease associated with hepatitis C. Such lesions are associated with more rapid
fibrosis progression and a less favorable response to treatment. The major development over
the last ten years, spurred by French teams, of non-invasive assessment of fibrosis during
the course of hepatitis C has significantly reduced indications for liver biopsy in patients
with chronic hepatitis C. Several serum tests (FibroTest, FibroMeter, Hepascore) are
currently being validated by the French High Authority of Health for establishing extent of
fibrosis in patients with untreated chronic hepatitis C and no co-morbidity [3-5]. The FibroT
est has been validated by numerous studies and several independent teams [6]. The FibroM
eter virus [9] has also been the subject of independent validations by different teams.
FibroScan is useful for confirming or ruling out the presence of cirrhosis [7, 8], and for
patients with HIV-HCV co-infection. In a recent survey, the use of liver biopsy was reduced
by 50 % for chronic hepatitis C patients [9]. Most hepatologists in France no longer recom
105
106
mend first-line liver biopsy for chronic hepatitis C (Table 4). Discrepancy between results of
serum fibrosis markers and FibroScan, when performed simultaneously, is an indication
for liver biopsy.
Transparietal liver biopsy
Transjugular liver biopsy
Absolute contraindications:
Hydatid cyst
Absence of patient cooperation
Cholangitis
Clotting abnormalities (see text)
Bile duct dilatation
Need to maintain anticoagulants or antiplatelets
Uncorrected hemostasis deficits
Vascular lesion along the puncture route

Non-percussive or non-identifiable liver
Hydatid cyst
Suspicion of amyloidosis
Relative contraindications
Morbid obesity
Ascites
Infection of the right pleural cavity
Table 4. Contraindications to transjugular and transparietal liver biopsy
2.2. Hepatitis B and hepatitis B-delta

Serum markers of non-invasive necroinflammatory lesions and fibrosis in hepatitis B and Bdelta have not been fully validated, nor has the FibroScan [10, 11]. Scientific institutions rec
ommend that liver biopsy be performed prior to any treatment decision in the context of
chronic hepatitis B or B-delta. Liver biopsy is the best means of assessing necroinflammatory
lesions and fibrosis in chronic hepatitis B [12]. The evolutionary trend in indications for liver
biopsy for hepatitis C and B has been inverted over the last twelve years: the number of liver
biopsies for hepatitis C in 2009 represented 33.6%, compared to 54.1% in 1997, this trend be
ing related to development of non-invasive measures for assessing fibrosis; however, the
number of liver biopsies for hepatitis B and delta-B has tripled in France [13] (Table 4). This
is probably related to the increased number of patients with hepatitis B in France and the
emergence of more effective treatment, along with insufficient validation of non-invasive
fibrosis assessment methods.
2.3. Alcoholic liver disease
Liver biopsy remains essential in case of severe acute alcoholic hepatitis with Maddrey func
tion above 32. In this situation, lesions of acute alcoholic hepatitis are absent in 20% of cases
[14], while the benefit of corticosteroids in the absence of alcoholic hepatitis lesions has not
been demonstrated with an increased risk of bacterial infection. However, this point has
been debated by some authors [15]. When there are no signs of severe acute alcoholic hepati

http://dx.doi.org/10.5772/52618
tis and if extensive fibrosis or cirrhosis is suspected, then the Fibrotest [16]and FibroScan
[17] give satisfactory diagnostic performances.
2.4. Non alcoholic fatty liver disease
In patients with hepatic steatosis as part of the metabolic syndrome, liver biopsy is useful
for differentiating fatty lesions from steatohepatitis (NASH), the evolutionary potential of
which is much more severe (risk of cirrhosis and hepatocellular carcinoma ). The presence of
body mass index > 30 kg/m, AST/ALT ratio > 1, hypertriglyceridemia > 1.7 mmol/L, age > 50
years and a syndrome of insulin resistance are predictors of steatohepatitis and fibrotic le
sions. When elements of metabolic syndrome exist with or without steatosis visualized on
ultrasonography, then LB performed for what is referred to as unexplained" cytolysis leads
to a diagnosis of steatosis and steatohepatitis lesions in 60% of the cases [18]. Liver biopsy
enables accurate diagnosis of lesions and evaluation of the degree of fibrosis [1]. It should be
noted, however, that in this setting, steatosis FibroMeter [19] and Fibromax [20] can pro
vide evidence of the existence of fibrosis and can predict the existence of NASH.
2.5. Cholestatic liver diseases, and autoimmune diseases of the liver
Diagnosis of primary biliary cirrhosis is based on identification of cholestasis associated
with antimitochondrial M2 antibodies. Liver biopsy is not useful for diagnosis of primary
biliary cirrhosis [21], but is very useful for assessing the activity and extent of fibrotic le
sions. FibroScan in this indication can assess the presence or absence of cirrhosis [22]. Liver
biopsy is useful in case of a poor response to ursodeoxycholic acid and/or in case of a drastic
increase of transaminases. Liver biopsy is able to reveal moderate to severe lymphocytic
piecemeal necrosis that may fit into the context of overlap syndrome, requiring a change in
therapy and the addition of corticosteroids. During the course of autoimmune hepatitis [1,
23] , liver biopsy is necessary to assess piecemeal necrotic lesions and fibrosis stage. It is es
pecially helpful in the absence of antibodies. In autoimmune hepatitis, no method of noninvasive evaluation of fibrosis has been developed. Liver biopsy is also necessary prior to
discontinuation of immunosuppressive therapy, since the presence of histological piecemeal
necrotic lesions is associated with almost constant recurrence of outbreaks of cytolysis dele
terious to the liver [24]. When confronted with possible chronic cholestasis, the diagnosis of
primary sclerosing cholangitis is based on data from the magnetic resonance cholangiopan
creatography (MRCP)[21]. Liver biopsy often confirms the diagnosis, but can appear normal
in 25% of the cases. When MRCP is normal, liver biopsy enables diagnosis of cholangitis of
small bile ducts and, in all cases, helps to clarify lesions due to hepatic fibrosis [1, 21].
2.6. Genetic hemochromatosis
Diagnosis of hereditary HFE-gene-related hemochromatosis is based on the association of
hyperferritinemia with elevated saturation of transferrin and presence of the C282Y muta
tion in the homozygous state. Thus, liver biopsy is not mandatory for diagnosis. It is still
indicated, however, when serum ferritin is higher than 1,000 g/L, and/or when the AST
are increased and/or if hepatomegaly is present [25]. Simple markers (platelet count and
107
108
transaminases, possibly combined with the dosage of hyaluronic acid and/or use of Fibro
Scan) can indicate the existence or absence of extensive fibrosis and help to guide indica
tions for liver biopsy.
2.7. Unexplained abnormal liver tests
Liver biopsy is often proposed in case of unexplained abnormal liver tests, when physical
examination, biochemical and serological tests, imaging investigation could not establish a
diagnosis. In one study including 354 patients, non alcoholic fatty liver disease was the defi
nite diagnosis in 64 % of the cases. Other lesions included drug induced liver injury, alcoholrelated liver disease, auto-immune hepatitis, primary sclerosing cholangitis,primary and
secondary biliary cirrhosis, hemochromatosis, amyloid and glycogen storage disease, and
cryptogenic hepatitis [26]. In another study including 272 patients, NAFLD represented 59.5
% of the cases [18].
2.8. Other indications (Table 3)
Liver biopsy is essential for the diagnosis of rare diseases of the liver such as Wilson's dis
ease, wherein the hepatic copper concentration has to be measured, a deficiency in al
pha-1 antitrypsin with evidence of PAS-positive cells, overload diseases such as Gauchers
disease, and amyloidosis, when there exists no other alternative [2]. In case of amyloido
sis, liver biopsy should be performed via the transjugular route, since there is a major risk
of bleeding in case of LBP performed via the transparietal route. Liver biopsy also helps
in diagnosing rare diseases (nodular regenerative hyperplasia, congenital hepatic fibrosis)
in case of prolonged abnormal liver function tests [1]. In case of severe acute hepatitis,
emergency liver biopsy performed via the transjugular route may be particularly useful
for diagnosing seronegative autoimmune hepatitis, infiltrative lesions of the liver, hepati
tis or herpes [1]. Liver biopsy is essential for diagnosis of abnormalities in liver function
tests when monitoring patients after liver transplantation in order to give a positive differ
ential diagnosis of the following anomalies: rejection, infection, drug-induced liver injury,
bile duct injury and viral reinfection. In case of hepatitis C virus recurrence in the liver
transplant, liver biopsy is indicated; however, the FibroScan is currently being assessed
for evaluating damage from hepatic fibrosis. In case of suspected drug-induced hepatitis,
liver biopsy may be useful if biochemical abnormalities persist beyond 3 months after ces
sation of treatment or if there is evidence suggesting injury to the bile ducts, such as a
prolonged cholestatic syndrome.
It is essential that the pathologist be provided with relevant and complete clinical and bio
logical information. Such information should be available before performing liver biopsy in
suspected cases of rare diseases of the liver, or when bacteriological seeding or special stain
ing has to be performed [1], so that the fresh liver fragment is immediately transmitted to
the pathology or microbiology laboratory.

http://dx.doi.org/10.5772/52618
3. Limitations
Liver biopsy has remained the gold standard for years. However, it is imperfect since a large
biopsy is required to make an accurate assessment of fibrotic stage and inflammatory grade.
Pathologists estimated that a 25 mm-long fragment obtained with a 16-G needle was necessary
to accurately determine the grade of chronic liver disease [27]. Colloredo et al. showed that
eleven to fifteen complete portal tracts was the minimal number below which disease stage
was significantly underestimated [28]. In a large review of the literature including 10,027 LB,
Cholongitas et al. showed that the mean SD length was 17.75.8 mm and the mean SD num
ber of portal tract was 7.55.8 [29]. This implies that at least two passes would be necessary to
obtain a 2.5 cm long specimen, thus potentially increasing the risk of complications.
4. Optimal methods for carrying out lb in 2012

Methods for performing LB will not be detailed here, but are available in practical guide
lines [30]. Several issues will be addressed:
When all conditions are met, then "ambulatory" liver biopsy may be performed [30]. In the
study published in 2000 (completed in 1997) [31], 27% of liver biopsies were performed on
an outpatient basis, most often for chronic hepatitis C; this figure is currently at 45% [13].
Several French teams have shown that outpatient liver biopsy is a safe and effective proce
dure and that liver biopsy performed on an outpatient basis reduces discomfort and increas
es the acceptability of subsequent examination conducted under the same conditions. If all
conditions are not met and/or if organizational arrangements do not permit it, then liver bi
opsy should be performed via traditional hospitalization.
LB is carried out by hepatogastroenterologists, radiologists and occasionally by surgeons.
Currently, liver biopsy is performed in France by a hepatogastroenterologist in 63.5% of cas
es, by a radiologist in 34.8% of cases and by a surgeon in 1.7% of cases [13]. The increasing
number of liver biopsies performed by radiologists in France is linked to an increased num
ber of biopsies performed using ultrasound guidance or guided real-time ultrasonography
[30] and by the development of the transvenous route as compared to the transparietal
route. Indeed, in 1997, 9% of liver biopsies were performed via the transvenous route com
pared to 22.4% in 2009 [13]. In the US, 50% of biopsies are performed by radiologists. In that
country, it is felt that the number of LB performed in order to gain sufficient expertise is at
least 40, carried out in the presence of an experienced radiologist [1].
5. Absolute and relative contraindications for LB

Absolute and relative contraindications for LB depend on the surgical approach recom
mended. Contraindications for liver biopsy using the transjugular or transparietal route [1,
30] are summarized in Table 4.
109
110
5.1. Lack of patient cooperation

Absence of patient cooperation is an absolute contraindication for transparietal LB. Indeed, in
case of uncontrolled respiratory movements or agitation of the patient, the biopsy needle may
cause a tear in the liver capsule, bleeding or a pneumothorax. If the patient is unable to main
tain breath holding, or in the absence of expected cooperation, the question of the appropriate
ness of the indication for liver biopsy must be raised. If this indication is maintained, then liver
biopsy under general anesthesia might be necessary, eventually via the transvenous route.
5.2. Hemostasis disorders, history of unexplained bleeding, hemostatic disease
It is recommended that transparietal liver biopsy should not be performed if the prothrombin
rate is less than 50%, the platelet count is below 50 Giga/L or 60 Giga/L, if activated partial
thromboplastine time is greater than 1.5-fold that of the control or when bleeding time is
lengthened. The need for maintaining an anticoagulant or an antiplatelet is also a contraindica
tion to transparietal LB. Likewise, the existence along the puncture route of a hemangioma or a
vascular tumor is a contraindication for liver biopsy without real-time guidance. In this case,
ultrasound-guided liver biopsy may eventually be used [1]. For patients with hemophilia,
transvenous liver biopsy can be performed safely after correction of anomalies. In this indica
tion, non-invasive blood markers of fibrosis and FibroScan are particularly useful.
5.3. Impossibility of carrying out liver detection
The incapacity to detect the liver by percussion or ultrasound is an absolute contraindication
to performing transparietal liver biopsy, as is suspicion of hydatid cyst.
5.4. Dilatation of extrahepatic or cholangitic bile ducts
Dilatation of the extrahepatic bile ducts and cholangitis are contraindications to transparie
tal liver biopsy [1].
5.5. Relative contraindications
Morbid obesity, severe ascites persisting after evacuation and infection of the right pleural
cavity are contraindications for transparietal liver biopsy [1]. The transvenous route can be
used in all these settings, and especially in case of significant ascites, morbid obesity, vascu
lar liver, anticoagulant or antiplatelet treatment that cannot be stopped, hemodialysis,
chronic renal failure or suspicion of amyloidosis when liver biopsy is necessary. Contraindi
cations for the transvenous route include bacterial cholangitis, hydatic cyst and uncorrected
deficits in hemostasis [1, 30].
6. Reducing the risk of complications

Compliance with absolute and relative contraindications for liver biopsy should lead to a
decrease in serious accidents due to LB, the presentation of which is beyond the scope of this

http://dx.doi.org/10.5772/52618
paper. Liver biopsy is an invasive procedure with the possible risk of severe complications,
approximately 0.5/1,000 [1, 30]. Liver biopsy is a procedure for which there exists residual
mortality [32]. Although serious complications have decreased over time, mortality after
performing transparietal liver biopsy remains at 0.2% and deaths related to liver biopsy for
diffuse parenchymal liver amount to 1 out of 10,000 LB [32].This risk, however, has de
creased dramatically over time because of improvement in indications for liver biopsy and
compliance with contraindications [32].
7. Conclusion
Liver biopsy remains useful for making an etiological diagnosis and a prognostic evaluation
of many non-viral liver diseases, particularly in the context of autoimmune liver diseases, as
well as for monitoring liver transplant patients. Liver biopsy is of great value in cases of sev
eral associated parenchymal diseases, so as to determine the extent of each, especially in
hepatitis C. However, within the setting of isolated hepatitis C without co-morbidity, we
feel that first-line LB is no longer appropriate.
The authors declare that they have no conflicts of interest.
Author details
Jean-Franois Cadranel1 and Jean-Baptiste Nousbaum2
1 Service dHpato-Gastroentrologie et de Nutrition,Centre Hospitalier Lannec, France
2 Service dHpato-Gastroentrologie, Hpital de la Cavale Blanche, Brest, France
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[13] Cadranel JF, Nousbaum, Hanslik B. Major trends in liver biopsy practices in France:
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[14] O'Shea RS, Dasarathy S, McCullough AJ. Alcoholic liver disease. Hepatology. 2010;
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[30] Nousbaum JB, Cadranel JF, Bonnemaison G, Bourliere M, Chiche L, Chor H, et al.
[Clinical practice guidelines on the use of liver biopsy]. Gastroenterologie clinique et
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Chapter 7
Primary and Metastatic Tumours of the Liver:

Expanding Scope of Morphological and
Immunohistochemical Details in the Biopsy
Ilze Strumfa, Janis Vilmanis, Andrejs Vanags,
Ervins Vasko, Dzeina Sulte, Zane Simtniece,
Arnis Abolins and Janis Gardovskis
http://dx.doi.org/10.5772/52838
1. Introduction
Evaluation of liver biopsy for tumour diagnostics is a highly practical task with major clini
cal influence. The liver is frequently affected by wide spectrum of neoplasms including be
nign tumours as well as primary malignancies [1-3]. In addition, due to the rich dual blood
flow to liver, secondary malignant tumours also often develop here. In order to ensure the
optimal management of the patient, a correct diagnosis is necessary. At present, biopsy is
the gold standard in oncology [4-5].
The scope of liver neoplasms can be following. The benign tumours include hepatic adeno
ma, bile duct adenoma, cavernous haemangioma and angiomyolipoma, among others. The
primary liver malignancies embrace hepatocellular carcinoma [6,7], cholangiocarcinoma [3]
and hepatoblastoma [8]. The diagnostics of hepatocellular carcinoma (HCC) is especially ur
gent topic due to high incidence in Asia and rising in Europe and USA, possibly because
of high prevalence of chronic hepatitis C [4,9]. Also, prognostic data should be reported in
cluding the features of early vs. progressed HCC, presence of stem cell immunophenotype,
multicentric growth or metastatic spread [7]. Among mesenchymal malignant tumours, epi
thelioid haemangioendothelioma and angiosarcoma [10,11] are notable. Metastatic tumours
represent the bulk of malignancies in Western countries [2]. Cystic liver tumours include
biliary cystadenoma and biliary cystadenocarcinoma [12-14].
Most of the above mentioned neoplastic processes can be diagnosed in core biopsy. The key
aspects include the following. First, the biopsy must be representative regarding the biologi
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cal process and radiologically detected changes [15]. Further, the obtained tissue must be
subjected to adequate technological process. Innovations here allow shortening the turnover
time significantly. Next, the evaluation of morphology must be done searching for the char
acteristic traits of the above noticed tumours. However, due to the limited tissue amount in
the biopsy, the tumour architecture sometimes is difficult to identify embarrassing the dis
tinction between nodular hyperplastic process, benign tumour or low-grade malignancy. In
contrast, high-grade malignancies can show significant cytological atypia by few signs of
differentiation embarrassing the detection of histogenesis [6] and the distinction between
primary and metastatic tumour.
Immunohistochemical markers as glypican-3 [1], Hep Par 1 [3,6], CD10 [3], alpha-fetopro
tein [6] and TTF-1 [16] are useful in the HCC diagnostics. Alterations of CD31 and CD34positive endothelial cell network reflect vascular remodelling during hepatic carcinogenesis
[7]. Cytokeratin (CK) 19 and 7 are characteristic for cholangiocellular carcinoma [3]. In meta
stases, organospecific markers including CDX2, mammaglobin, nuclear expression of TTF-1
or presence of neuroendocrine markers can confirm extra-hepatic origin [17]. As colorectal,
breast, lung and neuroendocrine cancers are frequent cause of metastatic liver damage [2] high
diagnostic value of immunohistochemistry (IHC) can be expected. However, the exact detec
tion of histogenesis can be difficult with metastatic pancreatic or gastric tumours and highgrade malignancies. IHC is mandatory for the diagnostics of haematological neoplasms and
epithelioid haemangioendothelioma. Assessment of tumour biological potential can be done
by IHC, evaluating Ki-67, Cyclin D1, FOXJ1, stem cell markers, matrix metalloproteinases and
other markers [7-8,18-22]. Novel markers appear continuously as heat-shock protein 70 [23].
Nowadays, pathology is not any more purely descriptive but it is becoming more functional
and clinically relevant. The classic morphologic characteristics must be combined with inte
grated evaluation of neoplastic process in the liver, including histogenesis, grading, clonal
changes, type and extent of vascularisation, immunophenotype, heterogeneity, prediction of
treatment sensitivity and the clinical behaviour [7]. New technologies as proteomic profiling
and genomic marker analysis should be applied in the evaluation of liver tumours [4]. Mi
croRNA studies can lead to new findings in cancer pathogenesis and prediction of treatment
efficacy [24,25].
The aim of the following chapter is to describe morphological and immunohistochemical
characteristics of primary and secondary liver tumours in order to develop logistic basis for
differential diagnosis of these processes in biopsy materials. Short discussion about the gen
esis and clinical course of each tumour will be included as well.
2. Benign epithelial liver tumours

Liver cell adenoma and bile duct adenoma will be discussed here. The regenerative process
es with the emphasis on focal nodular hyperplasia are described considering the differential
diagnosis.
Primary and Metastatic Tumours of the Liver: Expanding Scope of Morphological and Immunohistochemical Details...
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2.1. Liver cell adenoma and its differential diagnosis with focal nodular hyperplasia
Liver cell adenoma or hepatic adenoma is defined as benign tumour arising from hepato
cytes. The epidemiology is characterised by female predominance (90%) and strong associa
tion with oral contraceptive use [26-27] as 85% of affected persons have such history. Liver
cell adenoma was rare before the era of oral contraceptives [27]. At present, the incidence
has increased but is still low: 3-4 /100 000 per year in long-term users of oral contraception
[27-29]. The patients mostly are 20-39 years old. The other risk factors of hepatic adenoma
include androgen burden. The tumours can also arise spontaneously or occasionally can be
related toglycogen storage diseases or diabetes mellitus. Clinically, the patients mostly are
symptomatic. Abdominal fullness can be attributed to the presence of mass lesion; pain can
be caused by necrosis [27]. Rupture and bleeding (40%) represent dangerous complications
[27,29-31]; the risk of these events is increased in pregnant ladies affected by liver cell adeno
ma due to prior use of hormonal contraceptives. Risk of malignant transformation also is
recognised [29,32]. By literature analysis, Farges and Dokmak concluded that 5% of resected
hepatic adenomas bear HCC foci [32]. The risk of malignant transformation is higher in ade
nomas exceeding the size of 5 cm irrespectively of the number of adenomas as well as in
males. Grossly, liver cell adenomas are mostly unifocal (80%) and subcapsular. The tumours
can be quite large (5-20 cm). In most cases (75%) adenomas are encapsulated [27]. However,
the capsule can be thin or absent [10]. In contrast to HCC, adenomas usually are not associ
ated with cirrhosis [31]. Otherwise, radiological similarities exist between adenoma and
HCC as both can be large, have rich vascularity and can undergo necrosis [31]. Microscopi
cally, the tumour is composed by hepatocytes lacking anaplasia and arranged in thin (1-2
cells) trabeculae [27,29]. Cellular atypia and macrotrabeculae must be absent. Single arterio
les, a pair of arteriole and venule or isolated biliary ducts are scattered throughout the le
sion. However, well-formed triads enveloped in connective tissue are absent within the
lesion. The tumour can be distinguished from normal liver by larger size of neoplastic cells,
presence of capsule and lack of triad-containing portal tracts. Steatosis, hydropic degenera
tion or Mallory hyaline can be observed. Fibrous tissue, haemosiderin and calcifications can
develop in the consequence of haemorrhage. The immunophenotype is characterised by ex
pression of Hep Par 1 and other antigens that confirms the hepatic origin and by lower pro
liferation than in HCC. Molecular typing is emerging for liver cell adenoma as well. At
present, up to 4 molecular types are identified:
1.
hepatic adenoma with TCF1 gene inactivation;
2.
inflammatory hepatic adenoma;
3.
beta-catenin-mutated non-inflammatory hepatic adenoma;
4.
hepatic adenoma not displaying any before described feature or unsuitable for analysis [29].
The hepatic adenomas with TCF1 gene mutation comprise 35-40% of liver cell adenomas.
The patients are female. The tumour loses the expression and functions of hepatocyte nu
clear factor 1 (HNF1) encoded by TCF1 gene. Inactivation of the gene can be caused by
mutation in both alleles or by combination of a mutation and 12q deletion leading to loss
of heterozygosity in the corresponding region [33]. Germ-line mutation of HNF1 gene man
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ifests as maturity-onset diabetes of the young (MODY), type 3, in association with liver
adenomatosis [34]. However, the spectrum of HNF1A somatic mutations in liver cell adeno
ma differs from that in patients with MODY3 and suggests genotoxic damage [35]. By IHC,
loss of liver fatty acid binding protein can be observed. Not surprisingly, the adenomas
show steatosis [29].
Inflammatory hepatic adenomas constitute 50% of liver cell adenomas and can be associated
with obesity, smoking and alcohol use. Pathogenetically, inflammatory hepatic adenoma is
characterised by IL-6 pathway activation centred on gp130 protein in IL-6 receptor. The recep
tor can be subjected to ligand-independent activation due to mutation in IL6ST gene, or the
levels of gp130 can be elevated. The IL-6 receptor activation leads to recruitment of inflamma
tory cells through gp130-mediated production of chemokine CCL20. The mutation was found
in 60% of inflammatory adenomas [36]. However, the IL-6 pathway activation is universal in
the inflammatory hepatic adenoma. Microscopically, inflammatory infiltrates are observed in
addition to the architecture and cytologic details of adenoma. Occasional bile ductules, dilat
ed sinusoids and arterioles can be present. Haemorrhage is frequent. By IHC, expression of
acute phase reactants serum amyloid A and C-reactive protein is marked [29].
A group of hepatic adenomas is associated with beta-catenin mutation [37-38].The beta-cate
nin pathway is not affected in TCF1 inactivated group [29,38]. Beta-catenin activation can be
assayed by immunohistochemical over-expression of glutamine synthetase or by aberrant
nuclear localisation of beta-catenin. However, the tumours can show dysplastic changes
more characteristic for HCC thus possibly this group will be reclassified into well-differenti
ated HCC [29,36].
The last group of hepatic adenomas (5%) lacking TCF1 inactivation, inflammatory signature
and beta-catenin mutation [29] could represent distinct group with peculiar pathway of mo
lecular pathogenesis or result of technological shortcomings.
The differential diagnosis of hepatic adenoma in biopsy includes low-grade HCC and hy
perplastic lesions like focal nodular hyperplasia, nodular regenerative hyperplasia and par
tial nodular transformation [27].
Focal nodular hyperplasia (FNH) is a comparatively frequent differential diagnosis of hepat
ic adenoma. The FNH incidence is estimated as 3% [29-30,39]. FNH is characterised by pres
ence of hypervascular stellate scar in liver parenchymal nodule. The blood vessels are
located in the middle of star-like fibrous tissue while the periphery is occupied by proliferat
ing bile ductules. The morphologically remarkable abundant vascularity is in accordance
with the hypothesis of the FNH origin due to microscopic arterial malformation [40-42]. The
crucial difference between FNH and adenoma is pathogenetic as the former is thought to be
hyperplastic lesion, while adenoma is a neoplasm. The presence of stellate scar and lack of
peripheral capsule in FNH contrasts with presence of peripheral capsule and almost com
plete lack of connective tissue or portal triads within adenoma. If the architecture is incom
pletely represented in the biopsy, molecular characteristics should be able to discriminate
between the two inherently different processes, the hyperplasia and tumour. The immuno
histochemical markers of biliary differentiation have been employed in the differential diag
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nostics between FNH and hepatic adenoma. As described by Walther and Jain, CK19 and
CD56 detect rich network of proliferating biliary ducts in the fibrous septa of FNH but re
veal only few isolated ducts within the parenchyma of hepatic adenoma. Expression of CK7
is remarkable for the focal presence in parenchyma of liver cell adenoma in contrast to FNH
while both lesions show expression of CK7 in biliary ducts. Thus, panel of CK19, CD56 and
CK7 can be advised to solve the differential diagnosis in core biopsy [29]. Immunohisto
chemical expression pattern of glutamine synthetase differs between normal liver tissue,
FNH and liver cell tumours as well. In healthy tissue, glutamine synthetase is present in per
ivenular hepatocytes. These positive areas are expanded in FNH [39]. In hepatic adenomas,
glutamine synthetase expression is either diffuse of negative. In the last situation, the nega
tivity in the tumour can be incomplete, with focally preserved expression in the tumour pe
riphery [29] and thus difficult to interpret, especially in small biopsies where the preserved
positive focus seems to be dominant.
In nodular regenerative hyperplasia, the liver contains many small regenerative nodules.
Partial nodular transformation affects hilar area and is characterised by group of regenera
tive nodules surrounded by fibrous tissue [27].
Considering the differential diagnosis with HCC, thick trabecular cords, cytologic anaplasia
and invasive growth reveal the malignant biological potential. The thickening of trabeculae
is defined as presence of more than 2 cell layers in the trabeculae. The anaplasia is recog
nised by nuclear hyperchromasia, prominent nucleoli and increase in the nucleo: cytoplas
mic ratio. Presence of mitoses practically excludes the diagnosis of hepatic adenoma.
Atypical mitoses are absolute evidence of malignancy. The invasive growth can manifest as
invasion through the capsule, infiltration into liver parenchyma and true invasion into
blood vessels [27].
2.2. Bile duct adenoma
Bile duct adenoma is defined as a benign neoplasm of portal bile ducts. The epidemiologic
data suggest rare occurrence. However, as the tumours mostly are small and asymptomatic
[27], the true incidence and prevalence is unknown. Grossly, bile duct adenomas are mostly
solitary (83%), subcapsular (95%) and small (below 1 cm). By light microscopy, the lesion is
characterised by demarcated proliferation of bile ducts lacking atypia. The immunopheno
type repeats the staining characteristics of biliary ducts exhibiting expression of cytokeratins
7 and 19 [27]. The differential diagnosis can include small foci of low-grade cholangiocarci
noma or metastatic low-grade adenocarcinoma, but the benign cytological appearance is
helpful. Von Meyenburg hamartoma differs from bile duct adenoma, as the hamartomas
would be multiple and show traits of cholestasis. However, the exact separation might not
be of crucial importance due to benign course of biliary adenoma and pathogenetic sugges
tion that biliary adenoma represent a reactive process rather than true neoplasm.
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3. Malignant epithelial primary liver tumours

Three primary liver tumours are of utmost importance. Hepatocellular carcinoma is the
most frequent primary epithelial liver tumour with grave prognosis. Cholangiocarcinoma
ranks second by the incidence except for endemic regions. Hepatoblastoma is notable for the
occurrence in the infancy.
3.1. Hepatocellular carcinoma
Hepatocellular carcinoma is defined as malignant tumour developing from hepatocytes
and/or showing hepatocellular differentiation. It is the most common primary malignant tu
mour of the liver constituting 80-85% of primary epithelial liver malignancies [29,43]. Con
sidering the epidemiology, the worldwide burden of hepatocellular carcinoma can reach 1
million of new cases per year. The incidence shows major geographic differences. HCC is
the 2nd most common cancer in Asia, and the 4th in Africa [10]. The annual age-standar
dised incidence is the highest in East Asia, including China and Japan. Low-risk areas com
prise Europe, esp. northern and western parts; North and South America, Australia and
New Zealand [10].The age-adjusted incidence rates in Mozambique are as high as 112.9 and
30.8/100 000 in males and females, respectively. In China these values reach 34.4 and 11.6. In
contrast, the age-adjusted incidence rates in British males and females are 1.6 and 0.8, re
spectively [31]. The HCC risk factors include liver cirrhosis independently of cause, chronic
hepatitis B or C, ethanol consumption and non-alcoholic liver steatosis as well as mycotox
ins. The aflatoxin B1 or other mycotoxins produced by Aspergillus fungi could be responsible
for part of HCC in areas where grains, rice and peanuts are stored in hot and humid condi
tions [31]. Most of HCC cases develop from dysplastic cirrhotic nodule [29], thus the differ
ential diagnostics between dysplastic nodule and cancer represent evaluation of one point in
a complex road of pathogenesis. Clinically, most of the patients approach doctor due to
symptoms attributable to mass lesion in the liver (abdominal pain, sensation of fullness), tu
mour-related intoxication (weight loss, weakness, lack of appetite) and loss of liver func
tions (jaundice). Alternatively, the symptoms can be related to pre-existing cirrhosis and the
tumour could be identified during routine control of cirrhotic patient or during workup for
unspecific or unrelated symptoms [31]. Radiologically, the number and size of tumour
masses can be evaluated. Ultrasonography can be used for screening. Typical findings re
garding vascularity include hypervascularity and thrombosis of portal vein, frequently due
to invasion. If it is necessary to confirm invasion into portal vein, biopsy can be obtained
from it [31].
By microscopy, the typical patterns include trabecular, acinar and ductular structure. The
neoplastic cells in low-grade cases resemble liver cells by possessing wide eosinophilic cyto
plasm and distinct cell borders. Nuclear atypia is present and nucleo: cytoplasmic ratio is
increased, although to different degree. Mitoses can be present; atypical mitoses can be ob
served (Figure 1). The architecture shows unequivocal deviations from normal structure
such as thick trabeculae with more than 2 cell layers (in contrast to adenoma), solid areas,
duct-like or gland-like structures. However, careful evaluation of the architecture under
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high power magnification must be carried out. There are many secondary phenomena rais
ing the similarity between HCC and liver tissue: presence of macrovesicular or microvesicu
lar fat, Mallory hyaline and bile. The capillaries can be dilated [27]. Among the
histochemical staining methods, absent reticulin staining [44] is characteristic. PAS stain can
reveal glycogen and intracytoplasmic globules; the latter structure remains positive after di
astase digestion [27,44]. With some experience, morphology is helpful to distinguish finely
granular glycogen or rounded globules in HCC from mucus droplets in metastatic adeno
carcinoma or cholangiocarcinoma.
Figure 1. Hepatocellular carcinoma displaying marked cytologic atypia. Note the presence of atypical mitosis. Haematox
ylin-eosin (HE), original magnification (OM) 100x.
Fibrolamellar hepatocellular carcinoma (FLHC) has distinctive aetiology, epidemiology and

course. The general HCC risk factors are not associated with this subtype [31]. FLHC is rare,
constituting only 1-4% of HCC [27]. It is less common in high-risk areas than in North
America and Europe. The patients are young adults or even children [10]. FLHC is diag
nosed at the mean age of 25 years in contrast to mean age of 52 years in typical HCC patient
group [27]. Controversial data are reported about the sex predilection: some but not all au
thors have noted that females are mostly affected [10,31]. Clinically, symptoms attributable
to liver enlargement, parenchymal damage (elevated liver enzyme level) or tumour-related
intoxication (weight loss or fever) can be present. Cirrhosis is absent. The tumour can be
multifocal, and metastases can affect lungs and regional lymph nodes [31]. The histological
structure is remarkable for the lamellar fibrosis. The stroma is composed of thick, parallel
strands of hyalinised collagen [27]. The cells are large, polygonal, with wide eosinophilic cy
toplasm. The vesicular nuclei possess large nucleoli. Cytoplasmic pale bodies are more fre
quent (up to 50% of cases) and more abundant than in other types [10,27]. The pale bodies
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are rounded and very lightly eosinophilic thus staining paler than the surrounding cyto
plasm. These structures represent cystically dilated endoplasmic reticulum. Pale bodies can
be positive for fibrinogen by IHC. The immunophenotype is remarkable for diffuse expres
sion of CK7. The hepatocellular differentiation can be confirmed by Hep Par 1; alpha-feto
protein is present in approximately 20% of cases. The FLHC prognosis is better than in the
general group. The mean survival is 32 months in contrast to 5.9 months in trabecular HCC
[27]. However, it is found that the beneficial prognosis of FLHC is different from cancer in
cirrhotic liver but not from HCC in the absence of liver cirrhosis [10].
IHC has an important role in the diagnostics of HCC. Frequently tested antigens include
glypican-3, Hep Par 1, alpha-fetoprotein, CD10, carcinoembryonic antigen CEA, TTF-1, argi
nase-1, evaluation of cytokeratins and endothelial network as well as MOC-31 and markers
of extra-hepatic tumours.
Glypican-3 is a cell surface protein [1] that is involved in the control of cell proliferation and
survival. Glypican-3-knockout mice exhibit alterations in Wnt signalling [45]. Glypican-3 al
so interacts with Hedgehog signalling pathway [46]. In the practical surgical pathology, the
value of glypican-3 is associated with the cancer diagnostics as it is expressed in 70-75%
HCC but not in benign liver tissue [48-49] or cholangiocellular carcinoma [1]. Hepatoblasto
ma can be positive as well. However, glypican-3 can be expressed in metastatic melanoma
[50], ovarian clear-cell carcinoma [51], choriocarcinoma, yolk sac tumour [52-53] as well as in
blastomas including neuroblastoma and Wilms tumour [54]. In addition, 10% of gastric can
cer cases are positive for glypican-3 [55]. In melanoma, 80% of tumours contain detectable
level of glypican-3 protein and mRNA [1]. Regarding ovarian cancer, the rate of glypican ex
pression could be as high as 18% of all ovarian cancer cases and 60% of clear cell carcinoma
cases [51]. However, negative reports regarding clear cell carcinoma of ovary are published
as well [53]. Glypican-3 is silenced in breast cancer, lung adenocarcinoma and mesothelioma
[56-58]. Another problem has been highlighted by Abdul-Al et al., who have described fre
quent granular cytoplasmic expression of glypican-3 in chronic active hepatitis C [59]. Re
generative changes were suggested as the explanation. Authors emphasized that
membranous staining was not observed in hepatitis [59]. Glypican-3 has prognostic signifi
cance in HCC as it is associated with poor prognosis [60] and shorter recurrence-free period
after liver transplantation [49]. The applications of glypican-3 could extend beyond liver bi
opsy and return to it. It could possible to use glypican-3 plasma levels for diagnostics and
monitoring of HCC [61-63]. Immunotherapy could be guided towards glypican-3; the
present research is exploring both antibody and cell-based immunological mechanisms
[64-65]. Cancer vaccine could be generated against this molecule [1]. Glypican-3 is among
genes that are distinctly expressed in liver cancer stem cells; it is suggested that glypican
could be promising candidate for gene therapy without inducing damage to normal liver
stem cells [66].
Hep Par 1 is positive in normal liver, liver adenomas and HCC. The antibody was devel
oped in 1993 using an immunogen from failed liver allograft. The target antigen has been
identified as carbamoyl phosphate synthetase. This enzyme catalyses the rate-limiting step
in the urea cycle and is located in the mitochondria [67]. The specifity and sensitivity of this
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marker in HCC diagnostics exceeds 80% and has reached 90% in several studies [6,67]. Un
fortunately, sensitivity is lower in high-grade HCC. The expression in non-hepatocellular tu
mours including colorectal, pancreatic, breast, urothelial, prostate cancer, neuroendocrine
tumours, renal cell carcinoma, melanoma and angiomyolipoma is either negative or focal.
However, few gastric, colorectal and lung adenocarcinomas can be positive [6,67]. In the bi
opsy material, heterogeneity in the HCC can cause diagnostic problems [6].
Arginase-1 is an enzyme involved in the urea cycle as well. It is found in benign hepatocytes
and hepatocellular neoplasms. The antibody has received high sensitivity estimates of 96%
and favourable performance characteristics [68,69].
Alpha-fetoprotein is an oncofetal protein produced by the liver and yolk sac endoderm. The
antigen is remarkable for expression in malignant hepatocellular tumours (Figure 2) in con
trast to benign liver tissue, and for the high specifity. However, sensitivity is low (30-50%)
and heterogeneity adds further problems in biopsy evaluation [6]. Nevertheless, positive ex
pression is valuable.
Figure 2. Heterogeneous intense cytoplasmic expression of alpha-fetoprotein in hepatocellular carcinoma. Immuno

peroxidase (IP), anti-alpha-fetoprotein, OM 100x.
Polyclonal antibodies against carcinoembryonic antigen (CEA) yield positive reaction more
than in 70% of HCC cases, while monoclonal anti-CEA only rarely stains HCC. Reactivi
ty with polyclonal CEA antibodies mostly is observed in canaliculi; this pattern can be
observed in benign or malignant liver tissues and is attributable to cross-reaction with
biliary glycoprotein on the canalicular surface [67]. The canalicular pattern is specific for
HCC and can be used to exclude cholangiocarcinoma and metastatic adenocarcinoma. It
is not useful in the differential diagnosis between HCC and benign hepatocellular mass
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lesions. Although good general sensitivity has been reported, it is higher in well or mod
erately differentiated HCC that present less problems regarding the differential diagno
sis with cholangiocellular carcinoma or metastasis [67]. Cytoplasmic stain is not observed
in healthy liver or benign neoplasms; it is characteristic of malignancy but seen mostly in
cholangiocellular carcinoma and metastatic neoplasms. The rate of cytokeratin fraction
expression is 15% for CK7, 20% for CK20 and 10% for CK19. Diffuse strong expression
of endothelial markers CD31 and CD34 is not characteristic for normal liver tissue in
contrast to HCC [27]. The visualisation of endothelial layer is valuable also in estimat
ing the thickness of trabeculae. However, pattern of diffuse, strong endothelial marker
expression has low sensitivity of 20-40%. The patchy expression is also difficult to evalu
ate in liver biopsies. The visualisation of endothelium thus is not recommended for the
distinction between adenoma and carcinoma [6].
The transcription factor TTF-1 is expressed as intense granular cytoplasmic staining in nor
mal liver parenchyma [16] and hepatocellular tumours (Figure 3). The reaction is ensured by
cross-reactivity with hepatocyte mitochondrial antigen and is seen with the clone 8G7G3/1
[69]. The reported sensitivity is 60-70%. However, it parallels the expression of Hep Par 1
decreasing the practical value [6]. Its expression can be retained even in metastatic HCC [16].
Figure 3. Granular cytoplasmic expression of TTF-1 in hepatocellular carcinoma. IP, Anti-TTF-1, OM 400x.
MOC-31 is an epithelial cell surface glycoprotein of unknown function. Evaluating liver bi

opsies, it is valuable as non-hepatocellular marker. MOC-31 is negative in HCC but positive
in most metastatic adenocarcinomas and cholangiocellular cancer [67]. However, mesothe
lioma is MOC-31 negative as well; calretinin should be used in the panel to exclude this pos
sibility [17].
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Molecular subtyping is emerging for HCC. The subtypes are distinguished by high prolifer
ation and chromosomal instability; by activation of Wnt signalling pathway and by interfer
on signalling due to tumour-infiltrating cells [70-77].
The requests for clinically relevant classification have resulted in the separation of HCC into
early and progressed entities. The early HCC is recognized as small (not exceeding the diam
eter of 2 cm), well differentiated and lacking vascular invasion. The invasion into portal tracts
can be present and is highlighted by lack of proliferating ductules. Macrovesicular steatosis
is present in 40% of early HCC but appears mostly in Eastern cohorts. It can be attributable to
incomplete neoarterialisation the process of portal tract replacement by unpaired arteries
outside the portal tracts. In early HCC, there is still comparatively large venous flow. The
tumours in general may be radiologically hypovascular. The early HCC is more likely to
become the biopsy target due to equivocal findings at imaging. Progressed HCC includes HCC
of higher grade (moderate or poor differentiation degree, G2 or G3), possessing vascular
invasion, larger size or stem/progenitor cell immunophenotype and mixed hepatobiliary
differentiation. The stem cell immunophenotype can be detected by IHC for CK19, EpCAM,
CD133, and mixed hepatobiliary immunophenotype by expression of CK7 and CK19 [7]. The
5-year survival is 89% in the early HCC group in contrast to 48% in the progressed group. The
intrahepatic metastatic spread must be distinguished from multifocal carcinoma that is prog
nostically better disease. The multifocal disease is characterised by nodule in nodule struc
ture or by presence of at least one G1 nodule [7].
The differential diagnosis includes benign hepatic lesions, metastatic malignancies and chol
angiocarcinoma. IHC is of major importance. Markers, that are expressed both in benign
and malignant liver cells (CEA by polyclonal antibody, CD10, Hep Par 1, TTF-1 and (occa
sionally) cytokeratins [27]) identify the hepatocellular origin of tumour but cannot be used
to prove the malignant biological potential of suspicious biopsied tissue. If these are found
in high-grade tumour, diagnosis of HCC is preferable in contrast to metastasis. The expres
sion of alpha-fetoprotein and glypican-3 is typical for malignant tumour of hepatocellular
origin [27]. These findings are important in differential diagnosis with non-hepatocellular
and/or metastatic tumour in line with other markers specific for particular histogenesis. Re
garding the differential diagnosis of HCC and dysplastic cirrhotic nodule, a panel of immu
nohistochemical stains is recommended employing glypican-3, glutamine synthetase and
heat-shock protein 70 [48,78-80]. In biopsy, the panel has lower sensitivity although good
specifity: accuracy 60.8% for 3 markers and 78.4% for 2 markers with 100% specifity. The
findings were acceptable even in the group of low-grade HCC: the accuracy still was 57%
for 3 markers and 72.9% for 2 markers with 100% specifity [23].
HCC (except fibrolamellar type) mostly is associated either with cirrhosis or chronic active
hepatitis with fibrosis that has not reached the degree of cirrhosis. To facilitate the differen
tial diagnosis between HCC and liver adenoma or FNH it is wise to take separate biopsies
from the lesion and from distant liver tissues if possible.
The future pathways for molecular diagnostics of HCC include mRNA analysis of GPC3,
survivin and LYVE1 genes [78]. Glypican-3, encoded by GPC3, and survivin is up-regulated
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in parenchymal HCC cells while LYVE1 protein is down regulated in endothelial cells in
case of malignancy. MYC pathway studies could also bring new information [29].
In addition, molecular studies can predict the HCC prognosis. Down-regulation of p57
accelerates the growth and invasion of HCC cells [18]. The reduced p57 expression corre
lates with larger tumour size, higher TNM stage, presence of extrahepatic metastases and
decreased survival. In cell lines, the down-regulation of p57 increases the expression of cyclin
D1 and CDK2, enhancing the cellular proliferation. The matrix metalloproteinase-1 (MMP-1)
and protease activated receptor-1 (PAR-1) are expressed in HCC but not in normal liver.
The up-regulation of MMP-1/PAR-1 axis has prognostic value [20] and potentially could be
used in the identification of malignancy. Co-expression of stem cell transcription factors
Oct4 and Nanog indicates aggressive tumour behaviour and predicts recurrence after HCC
resection [22]. FOXJ1 is over-expressed in HCC. It is associated with histological grade, poor
prognosis and with tumour cell proliferation [19]. Hedgehog signalling pathway mediates
invasion and metastasis of HCC via ERK pathway. Up-regulation of cell proliferation is
associated with down-regulation of p27 and p21 and up-regulation of cyclin D1 [81]. Osteo
pontin plays role in the proliferation of HCC through interaction with the cell surface recep
tor CD44 [82] and is considered the key mediator for vasculogenic mimicry [83]. Baxinteracting factor is over-expressed in HCC and correlates with shortened survival [84]. NYESO-1 protein is a potential marker for early recurrence after surgical treatment [85].
Hepatocyte nuclear factor 4 suppresses the HCC development [86]. Sulfatase 2 protects HCC
cells against apoptosis [87]. Interleukins as IL-17 and IL-6 have tumour-promoting role [88].
Interaction with matrix metalloproteinases 2 and 9 is likely [89]. Up-regulation of sirtuins
has been identified [90]. Typing of immune cells in biopsy is mostly done for research
purposes [91]. If any of those parameters will show prognostic and predictive value, the
relevant IHC analysis should be included in the protocol of liver biopsy evaluation. The
technological future developments include virtual microscopy. Fractal analysis [92] and
quantitative IHC can be applied [93].
Methylation studies have been carried out in HCC [94]. The expression of microRNAs is un
dergoing active analysis in HCC [95-96]. MicroRNAs are non-coding, short RNA molecules
that can bind to messenger RNA and to prevent their translation into protein, providing ad
ditional regulation of gene expression. MicroRNAs act as large-scale molecular switch due
to ability simultaneously down-regulate many genes. MicroRNA-181 down-regulates the
differentiation and maturation of hepatocytes [96]. Suppression of microRNA-181 expres
sion leads to reduced motility and invasion of HCC stem cells [25]. MicroRNA-182 could
promote metastasis [97]. MicroRNA-183 inhibits apoptosis [98]. MicroRNA expression can
be subjected to regulation with IL-6 [25]. Reduced expression of microRNA-26 in HCC is as
sociated with poor prognosis. However, better response of interferon alpha postoperative
adjuvant therapy can be expected [95]. MicroRNA-21 induces resistance to the anti-tumour
effect of interferon and fluorouracil combination therapy [99]. Circulating microRNAs are
valuable in tumour diagnosis and monitoring the treatment [24].
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3.2. Hepatoblastoma
Hepatoblastoma is defined as a primary malignant blastomatous liver tumour showing
complex differentiation towards fetal and embryonal hepatocytes as well as mature tissues
including osteoid, connective tissue and striated muscle. Epidemiologically, hepatoblastoma
is a rare malignant liver tumour of childhood with the incidence of 1 case / 1 million [8,10].
In children, hepatoblastoma is the most common primary liver tumour. Characteristically,
the tumour develops within first five years of life: 4% of hepatoblastomas are present at
birth, 69% have developed by 2 years of age and 90% - by 5 years of age. Only 3% of patients
are older than 15 years [100]. The risk of hepatoblastoma is increased in APC-mutation-car
rying children from familial adenomatous polyposis (FAP) kindreds. Clinically, enlarging
abdomen can be the first sign. The other possible manifestations include weight loss, ano
rexia, nausea, vomiting and abdominal pain. Jaundice is rarely observed [100]. Paraneoplas
tic syndromes can occur. Among those, anaemia and thrombocytosis are frequent.
Precocious puberty due to production of chorionic gonadotropin is rare. Grossly, the tu
mours mostly occur as single lesions [10] measuring 5-22 cm [100]. Pseudocapsule can de
velop due to compression of surrounding liver tissue. Microscopically, hepatoblastoma can
display any of different histological patterns, or combination of these patterns. The fetal epi
thelial differentiation is characterised by thin trabeculae of small cuboidal cells. The nuclei
are small and round with fine chromatin and small nucleolus. The cytoplasm can be either
clear or finely granular resulting in light and dark pattern under low magnification. Foci
of extramedullary haemopoesis can be present. The combined fetal and embryonal pattern
is characterised by presence of small tumour cells in solid or acinar groups. The small cells
have scant cytoplasm, higher nucleo: cytoplasmic ratio and coarse chromatin. Hepatoblasto
ma is called macrotrabecular if the cells compose 6-12 cell layers in the trabeculae in most of
the tumour. Larger cells are present in the macrotrabeculae in addition to fetal and embry
onal type. In teenagers, macrotrabecular hepatoblastoma must be differentiated from hepa
tocellular carcinoma. Small cell undifferentiated hepatoblastoma morphologically resembles
small cell cancer displaying solid small blue cell pattern with focal necrosis. Mixed epithelial
and mesenchymal hepatoblastomas contain mesenchymal components including fibrous tis
sue, osteoid, cartilage, striated muscle, bone or melanin [100]. Mixed epithelial and mesen
chymal hepatoblastoma with teratoid features is recognised by the presence of endodermal,
neuroectodermal and complex mesenchymal tissues. The neuroectodermal component can
comprise melanin, glial and neuronal cells [10].After treatment, connective tissue, necrosis
and signs of haemorrhage develop in association with residual neoplastic tissue, and squa
mous islands become more common. Immunohistochemically, expression of alpha-fetopro
tein, beta-catenin and cell cycle markers is associated with the histological pattern. The fetal
subtype is characterised by low proliferation that parallels the scant mitotic activity; alphafetoprotein can be present and the expression of beta-catenin is retained in the membranous
localisation. The combined fetal and embryonal subtype is characterised by shift of beta-cat
enin expression towards the nuclei in higher grade embryonal component. An interesting
circular pattern can be observed. In the rounded cell groups, the middle is occupied by pro
genitor-type pale, small cells displaying low proliferative activity and nuclear expression of
beta-catenin. The progenitor-type cells are surrounded by intensively proliferating embry
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onal type cells characterised by mixed nuclear and cytoplasmic expression of beta-catenin.
The outermost layer of these concentric structures is composed by fetal type cells with low
proliferative activity and retained membranous expression of beta-catenin. The small cell
subtype lacks alpha-fetoprotein but has high proliferative activity, usually reaching 80%; cy
tokeratins are expressed as well. Even in the mixed epithelial and mesenchymal hepatoblas
toma, cytokeratins and alpha-fetoprotein can be expressed even in the ostecyte-like and
osteoblast-like cells embedded in or associated with the osteoid, correspondingly [10]. In the
study of Purcell et al., cyclin D1 and Ki-67 were two markers (out of 5, including also betacatenin, E-cadherin and alpha-fetoprotein) that were shown to have prognostic value re
garding survival [8].
3.3. Cholangiocarcinoma
Cholangiocarcinoma (CC) is defined as malignant epithelial liver tumour with biliary histo
genesis or biliary differentiation. Epidemiologically, CC is a rare tumour with male predilec
tion. It composes 15% of primary liver cancer [100] but the relative incidence range of
cholangiocarcinoma is wide, from 5% in males and 12% in females in Osaka, Japan, to 90%
in males and 94% of primary liver cancer cases in females in Thailand. The age-standardized
incidence per 100 000 males ranges from 84.6 in Thailand to 2.8 in Osaka, Japan; 1.0 in
France or 0.9 in Italy. The known risk factors include association with ulcerative colitis and
primary sclerosing cholangitis [27]. The rate of cholangiocarcinoma in primary sclerosing
cholangitis patients is estimated as 10-20%. The presence of parasites, especially Clonorchis
sinensis and Opisthorchis viverrini, also increases the risk of cholangiocarcinoma. The high-in
cidence area in Laos and North and Northeast Thailand corresponds to the endemic area of
Opisthorchis viverrini. Korea has high rate of cholangiocellular cancer due to endemic spread
of Clonorchis sinensis. Clinically, the patients can present with painless jaundice [31], general
malaise, mild abdominal pain and weight loss [100]. Grossly, several types exist. Peripheral
tumours arise from portal bile ducts. Hilar lesions arise in large ducts. The diffuse intraduc
tal papillomatosis involves ducts as widespread carcinoma in situ lacking dominant mass
but leading to severe obstruction of bile flow. Histologically, cholangiocarcinoma has adeno
carcinomatous structure characterised by tubular complexes and moderate amount of des
moplastic stroma. The architectural variants include high-grade tumour lacking the
characteristic architecture, signet-ring cell tumour with presence of signet-ring cells, muci
nous type with extensive secretion of extracellular mucin, adenosquamous type with focal
squamous differentiation and spindle cell type with pseudosarcomatoid structure, presence
of malignant spindle cells and signs of epithelial differentiation. The tumour has no func
tional connection with bile excretory system although morphological connection in the form
of invasion or cancer in situ can exist. CC arises from ductal epithelium and not from hepa
tocytes. Due to these two reasons, presence of bile in the lumina of malignant glands is not
characteristic but eosinophilic or mucinous secretion can be present. Mucin stains as PAS or
mucicarmine can be positive [44]. The immunophenotype is derived from the immunophe
notype of bile duct epithelium, with expression of following cytokeratins: CK19 (100%), CK7
(80-100%), CK20 (20%). Diffuse cytoplasmic expression of CEA is found by polyclonal anti
body in almost all cases and is frequent by monoclonal antibody as well [27]. However, it is
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suggested that morphology cannot reliably distinguish cholangiocarcinoma from metastatic

pancreatic or colorectal cancer [31]. In case of pancreatic adenocarcinoma, the marked cellu
lar atypia disproportionally to better preserved architecture can be a clue. Colorectal adeno
carcinoma in typical cases is characterised by columnar morphology and diffuse intense
expression of CK20, CDX2 and CEA and lack of CK7. Other authors have drawn attention to
the impossibility to distinguish cholangiocarcinoma from metastatic gastric cancer and can
cer of gall bladder; metastatic pancreatic cancer also remains a problem [6]. The morphologi
cal differential diagnosis includes benign proliferation of bile ducts, hepatocellular
carcinoma and metastatic adenocarcinoma [27]. In order to discriminate between biliary ad
enoma and cholangiocarcinoma, invasion (including single invasive cells and perineural in
vasion) and cellular atypia should be sought for. Radiologic findings are helpful as bile duct
adenoma usually is smaller than 1 cm, but cholangiocarcinomas are large. The differential
diagnosis with hepatocellular carcinoma can rely both on morphology and immunopheno
type. Immunohistochemically, markers of biliary differentiation CK7 and CK19 are positive
in cholangiocellular carcinoma. Hep Par 1 can be used to exclude hepatocellular differentia
tion [6,29]. Proteomic analysis of differentially expressed proteins in peripheral cholangio
carcinoma is under research [101].
4. Vascular tumours
Cavernous haemangioma, epithelioid haemangiendothelioma and angiosarcoma are endo
thelial tumours representing the whole spectrum of biological potential. Haemangioma is
entirely benign although can cause complications due to large size; epithelioid haeman
gioendothelioma is notable for the peculiar structure leading to marked difficulties in the bi
opsy diagnostics, and angiosarcoma is a frank malignancy with grave prognosis. In
addition, angiomyolipoma will be discussed as well although it should be noted that this tu
mour has complex structure including rich vascularity as one component.
4.1. Cavernous haemangioma
Haemangioma is defined as benign endothelial tumour [102]. Due to bleeding risk, it is only
rarely seen in liver biopsy; in addition, the possibilities of radiological diagnostics are good
and the prognosis only rarely necessitates active treatment. However, epidemiologically the
lesion is the most common benign tumour of the liver with incidence 0.4% [27]. Clinically,
haemangioma usually are asymptomatic due to small size and slow expansive growth. Oc
casionally, a giant haemangioma (10-30 cm) can cause pain due to mass effect. Thrombosis
and bleeding can be dangerous complications. In neonates, blood shunting can lead to heart
failure. Grossly, haemangiomas are mostly solitary (90%), of small or moderate size (less
than 5 cm) and subcapsular. Microscopic structure is similar to cavernous haemangioma
elsewhere in the body. Cavernous, lake-like blood spaces can be seen, separated by hypocel
lular fibrous septa (Figure 4). Thrombosis can be present. The immunophenotype reflects
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the endothelial origin. In the rare situation, when biopsy is obtained from cavernous hae
mangioma, the differential diagnosis can include hepatic tumours with rich vascularity as
adenoma and cholangiocellular carcinoma. These are diagnosed by the presence and cyto
logical properties of liver cells. Other vascular tumours could be considered, including in
fantile haemangioendothelioma, angiomyolipoma, epithelioid haemangioendothelioma and
angiosarcoma. The infantile haemangioendothelioma can be recognized by capillary struc
ture and occurrence in infants [27]. Angiomyolipoma shows combination of fat, smooth
muscle and blood vessels with radiating immature smooth muscle cells. The higher cellular
ity and presence of fat are features incompatible with cavernous haemangioma. Epithelioid
haemangioendothelioma is discussed separately; the occurrence of vascular lakes usually is
not observed. Angiosarcoma can have cavernous architecture but the hallmark of it is the
cellular atypia.
Figure 4. Cavernous haemangioma in liver tissue. Note the large, cavernous spaces filled with red blood cells. HE,
OM 50x.
4.2. Angiomyolipoma
Angiomyolipoma is defined as benign mesenchymal tumour with complex structure includ
ing immature smooth muscle, blood vessels and fat. Epithelioid cells and perivascular
HMB-45-positive cells can be present. Research of the tumour histogenesis has resulted in
the concept of PEComa, a tumour of perivascular epithelioid cells, showing myomatous, lip
omatous and melaninogenic differentiation. Epidemiologically, liver angiomyolipoma is
rare. It has been diagnosed in wide age range (10-86 years). In tuberous sclerosis, the inci
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dence of angiomyolipoma is increased. These patients may develop multiple angiomyolipo

mas in liver as well as kidney angiomyolipoma. Awareness of this condition is necessary to
escape over-diagnosis of metastatic malignant tumour. Clinically, the tumour can be asymp
tomatic. However, large tumours can cause pain; rupture and bleeding is also possible. By
radiologic studies, the tumour is hypervascular again. Grossly, angiomyolipoma usually is
solitary (except in tuberous sclerosis), measuring 0.8-36 cm. The microscopic picture (Figure
5) is straightforward if all three components are present in liver biopsy and have typical
structure. The smooth muscle cells can have epithelioid appearance leading to morphologi
cal similarity to liver parenchymal cells; the rich vascularity could lead to diagnostic confu
sion with hepatocellular tumour already earlier. The epithelioid cells sometimes can cause
suspicion for malignancy due to large nuclei and nucleoli. However, the nucleo: cytoplasmic
ratio remains low due to increased cell size.
Figure 5. The microscopic structure of angiomyolipoma. Note the peculiar, thick-walled blood vessels, immature
smooth muscle proliferation with high cellularity as well as the presence of fat. HE, OM 100x.
In difficult cases, IHC is helpful. The smooth muscle cells express actin (Figure 6) and fat
cells S-100 protein. HMB-45 expression can be observed in perivascular epithelioid cells
(Figure 7). The differential diagnosis can include hepatocellular neoplasms or spindle cell
sarcomas. Actin expression and complex histological structure helps to exclude hepatocellu
lar origin of the tumour. Complex structure, combined immunophenotype and low prolifer
ation help to exclude sarcoma [27].
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Figure 6. Actin-positive smooth muscle component in angiomyolipoma. IP, anti-actin, OM 100x.
Figure 7. Expression of melanosome protein HMB-45 in angiomyolipoma. IP, anti-HMB-45, OM 400x.
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4.3. Epithelioid haemangioendothelioma

Epithelioid haemangioendothelioma (EHE) is defined as intermediate-grade malignancy de
rived from endothelial cells. The mean age of patients is 47 years, ranging 12-86 years. The
clinical picture can include symptoms related to enlarging mass in liver (abdominal pain,
hepatomegaly) and tumour-related intoxication (fatigue, malaise, anorexia). Radiologically,
the tumour can be found by computed tomography. EHE can be radiologically avascular
[10,27,103]. This finding is probably related to fibrosis and scarcity of functioning blood ves
sels despite the endothelial origin of the tumour. Grossly, multiple tumours can involve liv
er or liver and lungs. In the lungs, epithelioid haemangioendothelioma is known also as
intravascular bronchioloalveolar tumour. Despite the multifocality (Figure 8), slow progress
is possible in our experience.
Biopsy material is usually sufficient to diagnose the tumour. However, in our experience,
immunohistochemical investigation is crucial in order to find out the presence of tumour
cells on the background of stromal fibrosis and reactive inflammation, to detect the endothe
lial origin and to evaluate the low biological potential as reflected by low to moderate prolif
eration activity by Ki-67 (Figures 9-11).
Figure 8. Multiple foci of epithelioid haemangioendothelioma in liver biopsy. The tumour is highlighted by immuno
histochemical visualisation of vimentin regarding its mesenchymal nature. IP, anti-vimentin, OM 50x.
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Figure 9. Epithelioid haemangioendothelioma presenting as a fibrotic focus in liver biopsy. HE, OM 100x.
Figure 10. Loss of liver parenchyma due to infiltration of epithelioid haemangioendothelioma. IP, anti-cytokeratins
AE1/AE3, OM 200x.
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Figure 11. Expression of CD34 in epithelioid haemangioendothelioma. Note also the positive reaction in the lining of
a venule. IP,anti-CD34, OM 400x.
The tumour is growing within sinusoids and venules compressing the adjacent parenchyma.
As was mentioned, the expression of endothelial markers is typical. Focal expression of cy
tokeratin and/or actin is possible [103] and should not cause confusion if panel of immunos
tains is performed. Stromal fibrosis follows than and can become marked so that neoplastic
cells are obscured (Figure 9). Two cell types are described: epithelioid and dendritic. The
morphological differential diagnosis includes non-neoplastic fibrosis and/or inflammation
and granulation tissue, angiosarcoma and metastatic cancers with marked stromal fibrosis.
The non-neoplastic conditions can be ruled out by tumour architecture as revealed by im
munohistochemistry. Epithelial tumours can be excluded by the predominance of endothe
lial markers by IHC. Among the vascular malignancies, the diagnosis of epithelioid
haemangioendothelioma is preferred for lesions with low grade atypia, absence of frankly
malignant spindle cells, low proliferation, limited destruction of surrounding liver tissue
and absence of necrosis.
4.4. Angiosarcoma
Angiosarcoma is defined as malignant tumour of endothelial cells. Epidemiologically, it is
characterised by rare occurrence in the liver constituting 2% of primary hepatic malignan
cies [11]. Elderly (50-60-year-old) males represent the largest group of affected patients [27].
The described risk factors include history of thorotrast use for arteriography, exposure to vi
nyl chloride in the plastics industry where it has been used for polymerisation, arsenic com
pounds (used as insecticides, possibly present in wine and used in the treatment of
psoriasis), copper compounds, pesticides and other chemical carcinogens. In all cases, long
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latent period (6-35 years) embarrass the data collection. The clinical picture can show signs
and symptoms of liver damage (hepatomegaly, local pain, jaundice), disorders of blood cell
function (anaemia, thrombocytopenia, disseminated intravascular coagulation), and tu
mour-related intoxication manifesting as weight loss. Ascites, bleeding into abdominal cavi
ty and liver failure is possible [27]. Grossly, multiple masses with signs of haemorrhage are
present. Morphologically, the cellular atypia as well as vascular differentiation can be ob
served in variable extent. High-grade tumours exhibit solid growth with few vascular
spaces. Immunohistochemically, endothelial markers CD31 and CD34 are expressed. How
ever, the immunophenotype can be not straightforward. In our experience, it is important to
use several endothelial markers. At first, the reactivity can be uneven [27]. Even more, CD34
is technologically beneficial antibody characterised with high affinity. However, during the
evaluation it is necessary to consider CD34 expression in non-endothelial tumours including
gastrointestinal stromal tumour and solitary fibrous tumour, among others.
5. Metastatic liver tumours

In Western countries, metastatic tumours represent the most common malignant liver le
sion with the rate 94-98% among all malignant liver tumours [27]. Almost all malignant
tumours, including carcinomas, sarcomas, melanomas and haematological malignancies, can
secondary involve the liver by haematogenous, lymphogeneous or transperitoneal spread.
Theoretically, metastatic tumour retains the morphological characteristics of the primary
site. However, the balance between anaplasia and differentiation can shift towards anapla
sia in such degree that signs of differentiation towards specific tissue or cell type are hard
ly recognisable. Some tumours like squamous cell cancer and melanoma lack specifity
regarding the organ of origin. Even adenocarcinomas retain few specific features. There
fore the differential diagnostics between primary and secondary liver tumours represents a
complicated practical task. Clinical data can be absent if metastatic liver lesion presents as
cancer of unknown origin. The diagnosis can be reached by logical analysis of morpholo
gy, IHC and molecular data. If the establishment of exact histogenesis is unsuccessful, the
biopsy investigation should be directed towards the analysis of treatment possibilities. Path
ologist should comment in detail morphological and immunophenotype data that could
either prove or disregard any particular type of treatment.
In case of liver metastasis, the primary tumour most frequently is located in colon, pancreas,
stomach, breast, oesophagus, genitourinary organs [100, 103]. Lung cancer can metastasize
to liver as well [6]. Neuroendocrine tumours, even small, can give rise to hepatic metastases.
The clinical course in this case can be prolonged and occasionally characterised by carcinoid
syndrome including flushing, diarrhoea and palpitations.
The spectrum of metastatic tumours in liver biopsies depend on the frequency of differ
ent tumours, the biological properties of different neoplasms predicting the possibility of
metastatic spread to liver as well as by the medical paradigm considering the indications
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for liver biopsies. In the files of single university hospital, metastatic tumours constituted
45% of tumours or tumour-like liver lesions. Adenocarcinoma was the most frequent histo
logical type of metastases (65.5%) comprising metastases of colorectal (48.2%), pancreatic
(13.5%), breast (13%), gastric (6.2%), lung (4.5%) and oesophageal cancer (3.7%). Neuroen
docrine carcinomas were seen frequently (16%). Lymphoma constituted 0.4% of all tu
mours [2]. Metastases in cirrhotic liver were rare [2]. In another study, including 130 cases
of metastatic liver disease, gastrointestinal tract was found to be the most common pri
mary location (45.3%) of cancer metastasizing to liver followed by neuroendocrine tu
mours (10.7%) [104]. In children, neuroblastoma, nephroblastoma and rhabdomyosarcoma
are the most frequent source of metastases [103].
The spread to liver occurs in 5-10% of patients with Hodgkins lymphoma and 15-40% of
non-Hodgkins lymphoma cases at the time of diagnosis. Leukemias can involve the liver as
well. Grossly, large cell lymphoma can form masses similarly to carcinoma. In case of Hodg
kins disease, the size of nodules is variable. Leukemic infiltrate can be present without visi
ble mass lesion. Myeloid leukemias preferentially infiltrate sinusoids, lymphoid portal
tracts, but hairy cell leukemia can involve both portal tracts and sinusoids forming small
blood containing cavities, surrounded by neoplastic cells [103].
Malignant melanoma (Figures 12-14) is one of the greatest challenges in diagnostic surgical
pathology [105] due to amelanotic, clear cell, sarcomatoid, small cell, haemangiopericytoid,
signet-ring cell, myxoid, metaplastic and rhabdoid forms. The diagnosis largely depends on
IHC. Evaluating the intermediate filaments, melanoma expresses vimentin. Despite the re
ported concerns of cytokeratin expression in melanoma, this is rare event (3%) in formalinfixed tissues. Similarly, the expression of glial fibrillar acidic protein and actin is observed in
1% of melanomas [105]. Interspersed normal cells should be excluded from evaluation of cy
tokeratin and actin reactivity. Melanoma is characterised by nuclear and cytoplasmic expres
sion of S-100 protein in 97.4-98%. S-100 protein can be observed in carcinomas, histiocytic
neoplasms and malignant peripheral nerve sheath tumour, therefore melanoma-specific an
tibodies, e.g., HMB-45 and MART-1/Melan-A must be included in the panel. Melanoma can
express bcl-2, CD10, CD68, CD56, CD57, CD99, CD117 antigens leading to diagnostic confu
sion with lymphoma, renal cell cancer, hepatocellular cancer, GIST, seminoma and other ne
oplasms. Expression of Melan-A is found also in metastatic adrenocortical carcinoma
(50-60%) that can be recognised by inhibin expression in around 70% of cases [6]. S-100,
HMB-45, Melan-A and inhibin are absent from HCC [6].
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Figure 12. Diffuse sinusoidal spread of undifferentiated malignant tumour. By immunohistochemistry, metastatic
melanoma was revealed (see also Figure 13). HE, OM 400 x.
Figure 13. Intense perinuclear expression of melanosome protein HMB-45 in metastatic melanoma. IP, anti-HMB-45,
OM 400x.
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Figure 14. Lack of cytokeratins AE1/AE3 in metastatic melanoma. Note the unusual sinusoidal spread. IP, anti-AE1/
AE3, OM 400x.
Metastatic breast cancer expresses CK7 but not CK20. However, this immunophenotype is
shared by many adenocarcinomas. To identify the tumour as metastasis from breast primary
tumour, gross cystic disease fluid protein fraction-15 (GCDFP-15) and/or mammaglobin can
be detected. The specifity of GCDFP-15 is estimated as 99%, and the sensitivity ranges from
50 to 74%. Breast cancers of luminal molecular type express oestrogen (ER) and progester
one receptors (PR). Naturally, the expression of female steroid hormone receptors is shared
by ovarian and endometrial cancer. Nowadays the detection of ER and PR is routine in
breast cancer diagnostics but less experience is obtained with expression of hormone recep
tors in extra-genital carcinomas. The scientific studies report expression of ER in carcinoma
of lung, stomach and thyroid [105]. The cross-reactivity can be associated by certain anti
body clones. Also, HER-2 positive and triple negative molecular types of breast cancer are
more prone to develop visceral metastases. Thus, negative ER/PR expression cannot exclude
metastatic breast cancer, and positive findings should be interpreted with caution recognis
ing the possibility of metastatic ovarian or endometrial cancer and cross-reactivity or true
expression of hormone receptors in extra-genital tumour. ER/PR expression in lung or thy
roid tumour can be controlled by TTF-1 protein expression and/or evaluation for neuroen
docrine markers and calcitonin.
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Figure 15. Surfactant A in pulmonary adenocarcinoma. IP, anti-surfactant apoprotein A, OM 400x.
Adenocarcinoma, squamous cell cancer, small cell cancer and carcinoid are the most fre
quent lung neoplasms. Lung adenocarcinoma is characterised by expression of CK7 (100%)
and TTF-1 (60-75%). Expression of CK20 is rare. Cytokeratins 5/6 and 34betaE12 can be
present but are not dominant in comparison with CK7. Vimentin can be found in lung
adenocarcinomas. Nuclear expression of TTF-1 and/or cytoplasmic expression of surfac
tant apoprotein A (Figure 15) is an evidence of pulmonary origin. Small cell cancer express
es neuroendocrine markers and pan-cytokeratin. The expression of chromogranin A and CK
AE1/AE3 can be limited to perinuclear dot reactivity. Simultaneous detection of leukocyte
common antigen can be suggested to perform differential diagnosis with haematological
neoplasm. Nuclear expression of TTF-1 protein is frequently present (Figures 16-18). The
high proliferation fraction by Ki-67 is characteristic albeit unspecific. The immunopheno
type of squamous cell cancer is unspecific and characterised by cytoplasmic expression of
CK5/6 and CK 34betaE12 in association with strong nuclear reactivity with p63 protein. CK7
can be present but is not dominant. TTF-1 protein is absent. Carcinoid is characterised by
neuroendocrine differentiation and low proliferative activity. The TTF-1 expression is not
frequent [17,105-107].
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Figure 16. Small cell cancer. Note the salt-and-pepper chromatin and high mitotic activity. HE, OM 400x.
Figure 17. Granular cytoplasmic and perinuclear expression of chromogranin A in small cell cancer. IP, anti-chromog
ranin A, OM 400x.
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Figure 18. Nuclear TTF-1 expression in small cell cancer. IP, anti-chromogranin A, OM 400x.
Mesothelioma is characterised by expression of CK7, CK5/6, vimentin and calretinin (Figure

19). HBME-1 can be expressed as well but lacks specifity.
Figure 19. Nuclear and cytoplasmic expression of calretinin in epithelioid mesothelioma.IP, anti-calretinin, OM 400x.
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Metastatic colorectal carcinoma can be recognised by diffuse intensive cytoplasmic expres

sion of CK20 and nuclear expression of CDX2 [108]. Carcinoid of the midgut and hindgut
also are positive for CDX2 [109].
Neuroendocrine tumours are characterised by strong cytoplasmic expression of chromogra
nin A and synaptophysin and negativity for Hep Par 1 [6]. CD56 is considered to be the
most sensitive neuroendocrine marker. In our experience, it shows reliable performance in
small or compressed biopsies making it especially valuable tool for the evaluation of scant
tissue material. Occasional CD56 expression in HCC is described [6].
Renal cell carcinoma is characterised by negativity for Hep Par 1 and CEA expression (by
polyclonal anti-CEA antibody). Unfortunately, the rate of RCC expression decreases from
50-80% in primary clear cell renal carcinoma and 60-90% in papillary renal cell cancer to
20% in metastatic renal cell carcinoma. CD10 can be present both in HCC and renal cell
carcinoma. Although the pattern of expression is different this can be difficult to evaluate,
especially in core biopsy. PAX-2 is advised as marker of metastatic renal cell carcinoma with
the expression rate 70-80%. Expression of vimentin is more characteristic in clear cell re
nal carcinoma (60-70%) than in hepatocellular carcinoma; chromophobe renal cell carcino
ma also is negative [6].
As tumour heterogeneity remains a source of problems [69] and the immunophenotype can
be inherently complex and subjected to cross reactivity, we recommend wide IHC panels in
cluding several markers for HCC and cholangiocarcinoma as well as markers for metastatic
tumour, including the organospecific antigens (see Tables 1-2).
Tumour
Immunophenotype
Malignant melanoma
Vim + CK AE1 / AE3 S-100 + HMB-45 + MART-1 / Melan A +
Lung adenocarcinoma
CK7+ CK20- CK34betaE12-/+ TTF-1+ Surfactant apoprotein A +
Small cell cancer
CK AE1/AE3 + ChrA+ CD56 +TTF-1 +
Squamous cancer
CK34betaE12+ CK7-/+ CK20 p63+
NET
CK AE1/AE3 + ChrA+ TTF-1 +/ - (lung) or CDX2 + (midgut, hindgut)
Breast cancer
CK 7 + CK20 MG +/ ER +/ PR +/
Colorectal cancer
CK20+ CK7- CDX2+ TTF-1-
Table 1. The immunophenotype of selected malignant tumours. Abbreviations in the Table: Vim, vimentin; CK,
cytokeratin; TTF-1, thyroid transcription factor 1; ChrA, chromogranin A; NET, neuroendocrine tumour; MG,
mammaglobin; ER, oestrogen receptor; PR, progesterone receptor
Antigen
Valuable positive expression
Notes
Glypican-3
Hepatocellular carcinoma
Occasional positivity in non-hepatocellular tumour
Arginase-1
Sensitivity for hepatocellular carcinoma 96%

Normal liver tissue positive
Metastatic tumours rarely positive [69]
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Hep Par 1
Sensitivity for hepatocellular carcinoma around 50% [69]

Gastric carcinoma can be positive
AFP
Sensitivity for hepatocellular carcinoma around 15% [69]
CD10
Renal cell carcinoma
Negative in adrenal carcinoma
CK7
Cholangiocellular carcinoma
Positivity does not exclude hepatocellular carcinoma
Metastatic cancers
Valuable for primary evaluation of malignant tumour within

liver
CK17
Positive tumours as pancreatic cancer (58%), squamous
Metastatic cancers
carcinoma
(75%),
urothelial
carcinoma
(75%)
and
cholangiocellular carcinoma (35%) can be distinguished

from negative ones (gastric, colorectal, prostate, breast
cancer, hepatocellular carcinoma)
CK19
Positivity does not exclude hepatocellular carcinoma
Metastatic cancers
CK20
Metastatic colorectal cancer
Useful in conjuction with CK7 for initial grouping of cancers
CDX2
Metastatic colorectal cancer and NETs Heterogeneous focal expression in gastric and pancreatic
showing adenocarcinomatous structure

carcinomas
Mucinous ovarian cancers can be positive
Morules in endometrioid carcinoma are positive
Calretinin
Mesothelioma
Squamous carcinoma frequently positive
Adrenal cortical carcinoma

Sex
cord-stromal
tumours
of
the
genital tract
Surfactant
Lung
apoprotein A
experience possess high affinity and is
adenocarcinoma.
In
our Reactivity in thyroid cancer (43% in small group) has been
useful, if positive
TTF-1,
nuclear
expression
Metastatic
pulmonary
adenocarcinoma
reported
Heterogeneous expression has been observed [69]
(75%
of
Regarding pulmonary adenocarcinoma, less subjected to
non- heterogeneity-related evaluation problems than surfactant
mucinous type and 10% of mucinous apoprotein A

type), small cell cancer (pulmonary, Endometrial (17%) or breast (2.4%) cancer occasionally
50-90%; non-pulmonary, 44-80%) or positive [69]
thyroid
follicular
cancer
and
including
medullary
papillary,
but
not
anaplastic carcinoma [69]

TTF-1, cytoplasmic Hepatocellular carcinoma
Expression in benign liver parenchyma is present
expression:
Gastric or prostatic cancer can show cytoplasmic positivity
Chromogranin ANET
and
synaptophysin
http://dx.doi.org/10.5772/52838
CD56
Oestrogen
NET, cholangiocarcinoma
Other tumours can be positive
and Breast, ovarian or endometrial cancer, Non-gynaecologic cancers can be occasionally positive,
progesterone
endometrial stromal sarcoma
including lung cancer (4-15-67%)
GIST
CD34 is co-expressed in GISTs
Seminoma
PLAP is co-expressed in germ cell tumours
Breast cancer
High heterogeneity
receptors
CD117
Mammaglobin
Sensitivity for breast cancer 40-85%

Ovarian (17%), endometrial (40-70%) and endocervical
(30%) carcinoma can also be positive [69]
GCDFP-15
Breast cancer
Sensitivity for breast cancer 50-60%

High heterogeneity
Not associated with mammaglobin thus simultaneous
evaluation can be recommended [69]
PSA
Prostatic cancer
Negative in 5% high-grade prostate cancers. Reactivity in

few breast carcinomas and rectal NETs have been observed
[69]
Pax8
Thyroid cancer
Sensitivity for thyroid cancer 79-100% and for renal cancer
Female genital tract carcinomas
71-98%
Renal cell cancer
Breast cancer is negative. Positivity is useful to discriminate

breast cancer from ovarian or endometrial cancer
NETs can be positive
P63
Squamous cell cancer

Urothelial carcinoma
Table 2. Panel of antibodies for liver biopsy evaluation. Abbreviations in the Table: AFP, alpha-fetoprotein; CK,
cytokeratin; NET, neuroendocrine tumour; TTF-1, thyroid transcription factor 1; GIST, gastrointestinal stromal tumour;
PLAP, placental alkaline phosphatase; GCDFP-15, gross cystic disease fluid protein-15; PSA, prostate specific antigen
6. Cystic biliary tumours

The cystic biliary tumours are defined by cystic structure and development of / differentia
tion towards intrahepatic bile duct epithelium. The group includes malignant biliary cysta
denocarcinoma and benign biliary cystadenoma. Epidemiologically, cystic biliary tumours
represent rare entities, with incidence of biliary cystadenocarcinoma approximately 1/10
million (corresponding to 0.01/100 000) and of biliary cystadenoma 1/100 000 - 5/100 000
[110]. Biliary cystadenocarcinoma is diagnosed mostly at the age 50-60 years [100]. Biliary
cystadenoma is diagnosed in younger patients: mean age 40.6 (range 30-51) vs. 51.3 (range
41-63) years in biliary cystadenocarcinoma group [14]. In other studies even larger age dif
ference (17 years) is found between patients affected by benign and malignant cystic biliary
tumours, respectively [111-112]. Cystic biliary tumours are more common in women:
80-100% of biliary adenoma and 63-71.4% of biliary cystadenocarcinoma are described in fe
145
146
male [14]. The clinical picture reflects the presence of mass lesion and is dominated by ab
dominal pain [113]. The other manifestations and complications include jaundice,
cholangitis, tumour rupture [114], haemorrhage [115], compression of the portal or caval
veins with possible subsequent ascites [113], hemobilia [12] and mucobilia [116]. Notably,
the tumour can progress slowly [117] with the clinical history of biliary cystadenocarcinoma
as long as 10-15 years [112,118]. The long course is is in accordance with the low grade of
malignancy and gradual development of tumour through stages of increased epithelial pro
liferation, dysplasia, in situ cancer and, finally, invasive cancer. Thus, long anamnesis of
cystic hepatic mass does not exclude the possibility of malignant tumour and the need for
careful follow-up if the cyst is not removed by operation. Although biopsy can be consid
ered in cases with unclear differential diagnosis, it is not the first choice because of the fol
lowing considerations. First, simple liver cyst is the main differential diagnosis of cystic
biliary tumours. Although biliary cystadenocarcinoma is rare, liver cysts have high preva
lence being present in 2.5% of the population [119] and cannot be distinguished from cystic
biliary tumours on the basis of CA19-9 and CEA levels [14,114]. However, core biopsy is un
likely to yield sufficient tissue in case of simple cyst or cystadenoma; it also is not suitable
for the diagnostics of focal malignancy and rarely can lead to peritoneal carcinomatosis [13].
Therefore radiological diagnostics, especially computed tomography, is essential [117].
Grossly, biliary cystadenocarcinoma is multicystic. Internal mural nodules are irregularly
distributed in the walls. The tumour most frequently is located within the liver (83%). Extrahepatic bile ducts (13%) or the gall bladder (0.02%) has been affected by this tumour as well
[14]. The size of cystic biliary tumours (1.5-30 cm) is not helpful in the differential diagnos
tics between simple hepatic cyst and cystic biliary tumours; it also has no correlation with
malignant biological potential [120]. The metastatic spread of biliary cystadenocarcinoma
can affect the liver, regional lymph nodes in the hepatoduodenal ligament, lungs, pleura or
peritoneum [100]. Histologically, biliary cystadenocarcinoma is characterised by clear-cut
signs of malignancy: cellular atypia, particularly nuclear polymorphism, mitotic activity and
invasion into surrounding stroma. The tumour architecture is cystic and papillary. The be
nign counterpart of biliary cystadenocarcinoma, the biliary cystadenoma lacks the malig
nant features [100] and is composed by either mucinous or serous benign epithelium. Most
of cystic biliary tumours possess characteristic mesenchymal, ovariantype stroma. Hypo
thetically, these tumours arise from bile ducts proximal to the hilum of the liver and share
the cystic structure and presence of peculiar ovarian-type mesenchymal stroma with muci
nous cystic tumours of the pancreas and retroperitoneum, leading to the hypothesis that ec
topic ovarian stroma during embryogenesis can become incorporated along the biliary tree,
in the pancreas and retroperitoneal space and cause the proliferation of the adjacent epitheli
um by production of the hormones and growth factors [121]. Origin from intrahepatic peri
biliary glands [122] or from ectopic rests of primitive foregut sequestered in the liver [114]
has been hypothesised. Development from pluripotential stem cells is suggested on the ba
sis of the presence of albumin messenger RNA and biliary type cytokeratins in the tumour
cells [123]. Biliary cystadenocarcinoma without mesenchymal stroma more frequently arises
in males and carries poorer prognosis in comparison with the tumour possessing mesenchy
mal stroma [122]. By immunohistochemistry, increasing proliferative activity by Ki-67 ex
http://dx.doi.org/10.5772/52838
pression as well as increasing p53 protein expression from adenoma to carcinoma was
shown in biliary cystadenocarcinoma without ovarian-type stroma [124]. Expression of cyto
keratin (CK) 7 and absence of CK20, CEA, alpha-fetoprotein, calretinin, CD31 and chromog
ranin is described [125]. However, presence of CK20, although typical for colorectal cancer,
is described in cholangiocarcinoma, especially non-peripheral [126]. It might be expected in
biliary cystadenocarcinoma with growing awareness about this entity.
There is evidence showing that at least some cases of biliary cystadenocarcinoma originate
from pre-existing biliary cystadenoma. These data include the age difference between biliary
cystadenocarcinoma and biliary adenoma patients [14] as well as morphologic findings of
malignant transformation in a lesion with focally innocuous structure [127].
Radiologically, presence of internal septations allows excluding a simple cyst. Vascularity of
septa is characteristic for cystic biliary tumours [14] and is considered by some specialists to
be more reliable in distinguishing biliary cystadenoma from cyst than the simple presence of
septations [117]. Biliary cystadenoma is characterised by smooth and thin internal septa, but
presence of enhanced mural nodules in the outer wall or septa is the most important sign of
malignancy. Calcification is not frequent but has been found specific for malignancy by
some [14] but not all [119] authors as far as cystic biliary tumours are concerned. Size, num
ber of septations or location of the neoplasm does not help to differentiate between benign
or malignant cystic biliary tumours [14]. Some authors have postulated that preoperative
differentiation between biliary adenoma and cystadenocarcinoma by radiologic imaging is
not possible therefore liver resection should be performed for all cystic biliary tumours
[120]. This assumption is based on the experience that internal papillae with arterial en
hancement may be present in both tumours so that computed tomography and magnetic
resonance imaging yield overlapping data.
The clinical differential diagnosis of cystic liver lesions, entering the differential diagnosis of
biliary cystadenocarcinoma, include developmental, neoplastic, inflammatory and traumatic
lesions as simple bile duct cyst, polycystic liver disease, biliary hamartoma, cystically degen
erated cases of other primary or metastatic liver tumours, abscesses, hydatid cyst, extrap
ancreatic pseudocyst, hematoma and biloma [119,128].
7. Conclusions
In conclusion, wide variety of neoplastic processes can affect the liver. Most of non-cystic
tumours can be reliably diagnosed in liver biopsy. Several demographic and clinical data
should be submitted along with the liver biopsy. Patients age and presence or absence of
clinical symptoms must be known. If there is history of contraceptive use it should be report
ed. Radiological data have high relevance: the size, localisation in respect to liver capsule and
number of focal liver lesions should be known to the pathologist. The vascularity should be
described. Knowing these data, pathologist should evaluate the haematoxylin-eosin stained
specimen. Wide panel of immunohistochemical stains can be recommended than.
147
148
Author details
Ilze Strumfa1*, Janis Vilmanis2, Andrejs Vanags2*, Ervins Vasko3, Dzeina Sulte3,
Zane Simtniece1, Arnis Abolins1 and Janis Gardovskis2
*Address all correspondence to: ilze.strumfa@rsu.lv
1 Department of Pathology, Riga Stradins University, Riga, Latvia
2 Department of Surgery, Riga Stradins University, Riga, Latvia
3 Faculty of Medicine, Riga Stradins University, Riga, Latvia
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159
Chapter 8
Nonalcoholic Fatty Liver Disease: A Pathological View

Joaqun Cabezas, Marta Mayorga and Javier Crespo
http://dx.doi.org/10.5772/52622
1. Introduction
Nonalcoholic fatty liver disease (NAFLD) is the most common liver disorder of our times.
The spectrum of this disease goes from steatosis to non-alcoholic steatohepatitis (NASH),
cirrhosis and hepatocellular carcinoma. NAFLD can appear in the context of many condi
tions. Probably, NAFLD could be a component of metabolic syndrome, with its complete
phenotypic expression: insulin resistance, obesity, type 2 diabetes, hypertension, hypercho
lesterolemia, and hypertriglyceridemia.
The pathogenesis involves insulin resistance, hepatic fat deposition, increased oxidant
stress, apoptosis, inflammation and fibrosis. At present day, a new hormone has been dis
covered. Muscle cells products this new hormone, called irisin. Irisin can induce changes in
adipose tissue.
Diagnosis of NAFLD cannot be performed with a single test and it should be one of exclu
sion, as well.
Nowadays, there is not a single therapeutic intervention. The focus of management should
be treatment of the risk factors for NASH (insulin resistance, obesity). Principal methods
used for weight management are dietary modifications and life style changes. Then, phar
macotherapy may include insulin sensitizers, cholesterol-lowering agents, anti-obesity and
anti-oxidant agents. Morbid obese patients may benefit from surgical weight loss, reducing
the progression of NASH.
2. Definition
NAFLD definition [1] requires that there is evidence of hepatic steatosis, either by imaging
or by histology and there are no causes for secondary hepatic fat accumulation (Table 1).
162
NAFLD is usually associated with metabolic risk factors such as metabolic syndrome, obesi
ty, diabetes mellitus, and dyslipidaemia.
COMMON CAUSES OF SECONDARY HEPATIC STEATOSIS

Macrovesicularsteatosis
Excessive alcohol consumption.

Hepatitis C (genotype 3)
Wilsons disease.
Lipodistrophy
Starvation
Parenteral nutrition.
Abetalipoproteinemia.
Medication (amiodarone, methotrexate, tamoxifen, corticosteroids)
Microvesicularstatosis
Reyes syndrome.
Medications (valproate, anti-retroviral medicines)
Acutte fatty liver of pregnancy
HELLP syndrome
Inborn errors of metabolism (LCAT deficiency, cholesterol ester storage disease, Wolman disease)
Table 1. Causes of secondary fat accumulation.
NAFLD includes a constellation of histological findings that goes from steatosis, to necroin
flammation, called NASH and progression to advanced fibrosis and cirrhosis.
3. Epidemiology
NAFLD is becoming the leading cause of liver disease. One of the causes is the increasing of
obesity[2].
The incidence of NAFLD has been evaluated in a few number of studies, it ranges from
31-86 cases/1000 person-year in Japan to 29 cases per 100000 person-year in England [3, 4].
The prevalence of NAFLD is increasing. Recent studies presented in the Digestive Diseases
Week 2012 summarizes this increased prevalence over the last 20 years [5, 6]. Investigators
report an increasing in obesity. This increase is followed by a rising in steatosis and NASH,
the presence of steatosis among obese people has increased from 23% in the 80s, 43% in the

http://dx.doi.org/10.5772/52622
90s and finally to 60% nowadays [4]. Even in non-obese patients, the prevalence of steatosis
increased from 12%, to 27% and 36%, respectively [5].
In children/adolescents, over the last 20 years, obesity has increased from 11% to 21%, sus
pected NAFLD from 4% to 10, and the prevalence of altered aminotransferases among obese
adolescents has increased from 17% to 37% [6].
4. NAFLD and liver biopsy

Liver biopsy remains the gold standard for characterizing liver histology in patients with
NAFLD. However, it is expensive and carries some morbidity and very rare mortality risk.
Thus, it should be performed in those who would benefit the most from diagnostic, thera
peutic guidance, and prognostic perspectives.
The last guideline for NAFLD management recommends liver biopsy [1]: in patients who
are at risk to have steatohepatitis and advance fibrosis; theses patients could be identified by
the presence of metabolic syndrome and NAFLD fibrosis score; and a liver biopsy should be
considered in patients in whom other etiologies are suspected and cannot be excluded with
out a liver biopsy.
Liver biopsy allows confirming the diagnosis, evaluation and semiquantitation of necroin
flammatory lesions and fibrosis.
On the other hand, liver biopsy suffers from challenges. An adequate biopsy represents only
1/50000-1/65000 of the organ. Sampled area should be carefully chosen and sample length
must be enough, a least 15mm. This size can reduce sample error. Finally, experienced path
ologist is important to haver a greater yield of findings.
5. Histology of NAFLD
NAFLD represents a histopathologic spectrum ranging from steatosis alone, to necroinflam
mation, summarized as NASH; and progression to advanced fibrosis and cirrhosis.
The histologic characterization of NAFLD and NASH may include description of steatosis
and cell injury in addition to inflammation and fibrosis. Kleiner and Brunt [7] propose cate
gorizing the histologic changes when studying NAFLD as follows in table 2.
The main histological characteristic of NAFLD is the accumulation of fat in the form of tri
glyicerides within hepatocytes, lesion termed steatosis (Figure 1 and 2); this term is defined
by the guideline [1] as NAFL non-alcoholic fatty liver, where the risk of progression to cir
rhosis and liver failure is minimal. The presence of >5% steatoic hepatocytes in a liver biopsy
is accepted as the minimum criterion for thehistological diagnosis of NAFLD [8].
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CATEGORY
DEFINITION
No significant evidence of fatty liver disease.
Insufficientsteatosis for diagnosis of steatosis, without

other changes (ballooning, fibrosis) that would suggest
steatohepatitis.
Steatosis:
Steatosis without specific changes to suggest a form of
Steatosis with inflammation.
steatohepatitis. This category may include spotty lobular
Steatosis with nonspecific fibrosis
inflammation and/or mild degrees of fibrosis of uncertain

significance.
Steatohepatitis:
Form of steatoshepatitis most common in adults; defined
- zone 3 borderline steatohepatitis
as a zone 3 centered injury pattern that includes steatosis,

inflammation, ballooning injury, (often with Mallory-Denk
bodies) with or without fibrosis. Borderline
steatohepatitsis has some, but not all ofthe features that
would allow a diagnosis of steatohepatitis.
Zone 1, borderline pattern
Form of steatohepatitis that occurs mainly in young

children, characterized by zone 1-centered (portal
inflammation, portal-based fibrosis, zone 1 steatosis,
ballooning injury in zone 1 if present).
Cryptogenetic fibrosis/ cirrhosis
Presence of fibrosis (usually advanced) or cirrhosis, with

little ton o steatosis and no changes (ballooning, MalloryDenk bodies) that would suggest borderline or definite
steatohepatitis. Other explanations for fibrosis (besides
steatohepatitis) should be considered.
Table 2. Histologic Categorization of NAFLD [7].
Figure 1. Steatosis. Hematoxylin-eosin stain.

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Figure 2. Steatosis. Hematoxylin-eosin stain.
Steatosis in NAFLD is usually macrovesicular, which refers to hepatocytes with single large
intracytoplasmatic fat droplet or smaller well defined droplets displacing the nucleus to the
cell periphery. This macrovesicularsteatosis is usually present in a zone 3 or panacinar dis
tribution; it differs from zone 1 steatosis that is a common distribution in chronic hepatitis C.
Azonal steatosis is most often seen in biopsies with advanced fibrosis [9].
The extent of steatosis can be evaluated and classified semi-quantitative. The most reprodu
cible method follows the acinararchiqueture dividing the liver parenchyma in thirds and as
sessing percentage involvement bay steatoic hepatocytes [8] table 3.
STEATOSIS SEMI-QUANTIFICATION
Mild
0 33%
Moderate
33 66%
Severe
> 66%.
Table 3. Steatosis semi-quantification according to acinar architecture [8].
NASH, under this concept is the histology pattern of NAFLD, which is at risk of developing
advance fibrosis. The minimal criteria for the histopathological diagnosis of adult NASH in
clude steatosis, hepatocyte injury, usually in form of ballooning, and lobular inflammation,
typically localized in acinar zone 3 [10, 11].
The key feature for the diagnosis of NASH is the ballooning injury (Figures 3 and 4), and it
is considered a marker of apoptosis [12]. This type of cell injury is characterized by a cell
that becomes enlarged and the cytoplasm becomes irregularly clumped with optically clear,
nonvesiculated areas. Ballooned cells are seen most frequently in zone 3 near the hepatic
veins, and lose this localization, becoming portal inflammation more prominent when the
disease progresses and in severe cases. Immunostaining of hepatocyte keratins 8 and 18
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might help to identify ballooned hepatocytes [13]. Ballooned degeneration is difficult to di

agnose even by trained pathologist, for that reason it can show significant inter-observer
variation [14].
Figure 3. Hepatocyte ballooned. Hematoxylin-eosin stain.
Ballooning degeneration is associated with an increased liver-related mortality [15].
Figure 4. Ballooning hepatocyte. Hematoxylin-eosin stain.
Mallory-Denk Bodies (MDB), also known as Mallory bodies, are eosinophilic, ropey cyto
plasmatic inclusion bodies in the hepatocyte of patients with chronic liver disease. This type
of lesion contains abnormal cytokeratin 8 and 18 filaments that have been ubiquinated.
Mallory bodies have an importance in disease progression and it is suggested a possible
prognostic role in steatohepatitis [16]. In a recent study, the presence of MBD was signifi
cantly associated with liver-related mortality [15].
Both ballooning degeneration and MDB can trigger the development of apoptosis. Apoptot
ic (acidophil) bodies are common in NASH. They can be identified as rounded, eosinophilic
cytoplasmic fragments, which appear to be free within the sinusoids or surrounded by

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Kupffer or other inflammatory cells. Apoptosis has been validated as an accurate marker for
diagnosis of NASH based on immunochemistry in liver tissue [17].
Inflammatory infiltrates (Figure 5) can be seen in the hepatic acini/lobules or the portal tract.
Lobular inflammation is usually mild, consists of a mixed inflammatory cell infiltrate, com
posed of lymphocytes, some eosinophils, and a few neutrophils. Polymorphs can be ob
served around ballooned hepatocytes that are called satellitosis (Figure 6). Kuppfer cells
aggregates as lobular microgranulomas and lipogranulomas may appear [10]
Figure 5. Mononuclear inflammatory infiltration. Hematoxylin-eosin stain.
Figure 6. Polymorph around ballooned hepatocytes, satelitosis.
Hematoxylin-eosin stain.Portal chronic mononuclear cell inflammation in adult NASH is

common and mild. When portal inflammation is greater than lobular other aetiologies
should be ruled out, such as chronic hepatitis C [18]. On the other side, a greater portal in
flammation than lobular inflammation can be seen in successfully treated patients [19]. In a
large database of liver biopsies from the NASH Clinical Research Network, including adults
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and children, portal chronic inflammation was associated with clinical and histologic fea
tures of severity and advance disease [20].
Vascular alterations in NAFLD. Recent paper has focused the study of NASH in microves
sels of the liver [21]. This work has found an intraacinar branch of the hepatic artery in the
perivenular region in active steatohepatitis. This finding is important because it can lead to
confusion for a portal tract resulting in an equivocal diagnosis. Likewise, the presence of this
vessel correlates with higher stage of fibrosis.
Fibrosis in adult NASH usually starts in acinar zone 3 and has characteristic chicken wire
pattern due to deposition of collagen an other extracellular matrix fibres along the sinusoids
of zone 3 and around the hepatocytes (Figure 7 and 8). Portal fibrosis has been reported in
cases of morbid obesity-related NASH and in pediatric NASH. Fibrosis predicts clinical out
comes in NASH [22]. there was noter from this study that the progression of the fibrosis is
accompanied of steatosis reduction. Approximately 37% to 41% of patients with NAFLD
have fibrosis progression over 3 to 10 years [22, 23]. The higher rates of fibrosis progression
were related to: body mass index, diabetes and low initial fibrosis [22]. When periportal fib
rosis was not present, there was a 100% of negative predictive value in predicting liver-relat
ed outcomes [23]. Steatosis, inflammation, ballooning and Mallory hyaline were not
associated with liver-related mortality after adjusting for the presence of fibrosis [15]. The
inclusion of fibrosis explains why the recent classifications for NASH used by Younossi [15]
and Matteoni [16], independently correlated with liver-related mortality. This observation
shows the importance of fibrosis in NAFLD, patients with NASH and fibrosis portends a
higher risk of death [24].
Figure 7. Fibrosis pattern: around hepatocytes. Masson trichrome stain.

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Figure 8. Zone 3 fibrosis perivenular/pericellular. Masson trichrome stain.
Other histological lesions that may be seen in NASH include megamitochondria, glycogen
ated nuclei and iron deposition.
Megamitochondria (giant mitochondria) are round or needle-shaped, eosinophilic, intracy
toplasmatic inclusions more commonly observed in hepatocytes with microvesicularsteato
sis. This abnormal mitochondria is a result of injury from lipid peroxidation or represent an
adaptive change [25]. Glycogenated nuclei are vacuolated nuclei usually observed in peri
portal hepatocytes. Their presence is more frequent in non-alcoholic etiology and it is rare in
alcoholic injury [26].
Finally, hepatic siderosis might be seen in NAFLD. One study of 293 liver biopsies (34,5% of
patients with NAFLD) investigates the relationship between iron deposition and NAFLD
[27]. Stainable hepatic iron described three histological patterns: hepatocellular pattern, re
ticuloendothelial system cell RES - (mainly Kupffer cell) pattern and mixed. RES pattern
was associated with advanced fibrosis and higher histological features of portal inflamma
tion, ballooning and definite NASH [27].
6. Histologic scoring systems

NAFLD histologic criteria requires an accumulation of more than 5% of fat deposition,
mainly in form of triglycerides. NAFLD was first described by Ludwig and colleagues [28],
and since then several systems for grading and staging NAFLD have been proposed.
In 1999, Matteoni and colleagues characterized histologic subtypes that correlate with clini
cal outcomes [16] table 4. In 2005, NASH Clinical Research Network developed NAFLD
activity score (NAS) [8]. This score comprises four features evaluated semi-quantitatively:
steatosis, lobular inflammation, hepatocellular ballooning and fibrosis. Fibrosis was classi
fied separately table 5. When NAS is >5 sensitivity and specificity for definite NASH were
0,75 and 0,83, respectively. Finally, a recent classification for NAFLD has been proposed by
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Younossi and colleagues [15]. This classification includes the evaluation of these histologic
features: steatosis with centrilobular ballooning, and/or Mallory-Denk bodies of fibrosis
see table 6.
CLASSIFICATION OF NONALCOHOLIC LIVER FATTY LIVER DISESASE (NAFLD) BY SUBTYPE

NAFLD subtype
Pathology
Clinicopathologic correlation
Type 1
Simple steatosis alone
No NASH
Type 2
Statosis + lobular inflammation only
No NASH
Type 3
Steatosis + hepatocellular ballooning
NASH without fibrosis
Type 4
Steatosis, ballooning, Mallory bodies or fibrosis
NASH with fibrosis
Table 4. Classification of NAFLD by subtype [16].
CLASSIFICATION OF NONALCOHLIC FATTY LIVER DISEASE BY NAFLD CLINICAL RESEASCH NETWORK

Histologic finding
Score
Steatosis
0-3
Lobular inflammation
0-3
Hepatocellular ballooning
0-2
NASH requires a score of 4 with at least 1 point o ballooning injury.

Fibrosis type
Score
None
Perisinusoidal zone 3
Mild
1A
Moderate
1B
Portal/periportal
1C
Persinusoidal and portal/periportal
Bridging
Cirrhosis
Table 5. Classification of NAFLD by NAFLD CRN [8].

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CLASSIFICATION OF NAFLD BY SUBTYPE

Pathology
Clinicopathologic
correlation
Simple steatosis alone
No NASH
Steatosis + lobular inflammation only
No NASH
Steatosis with centrilobular ballooning and/or Mallory-Denk bodies
NASH
Any steatosis with centrilobularpericellular/perisinusoidal or briding fibrosis
NASH
Table 6. Classification of NAFLD proposed by Younossi and colleagues [15].
The most important difference between NAS and subtype classifications is that the latters
include fibrosis and this provides a better prediction of liver-related mortality in patients
with NAFLD [15].
7. NAFLD in special populations

Patients with insulin resistance. Insulin resistance can be estimated using the homeostasis
model for assessing of insulin resistance (HOMA-IR), calculated as the product of fasting in
sulin level (mUI/ml) and plasma glucose level (mmol/ml), divides by 22,5 [29]. Portal fibro
sis has been linked to the ductular reaction (ductularproliferation at the portal tract interface
arising from progenitor cells in the periportal area and accompanied by neutrophils and
stromal changes). These findings correlate the insulin resistance with advanced stages of fib
rosis and provide a pathway for fibrosis progression [30].
In some cases in a study with a few number of patients treated with an insulin sensitizer,
histologic evaluation of post-treatment liver biopsy showed that increased portal inflamma
tion is a feature related to resolution of NASH, and it is associated to a change in the quality
of zone 3 perisinusoidal fibrosis from dense to delicate [19].
NAFLD in bariatric surgery patients. Patients undergoing bariatric surgery for weight
loss are at a risk of NAFLD. They often have comorbidities such as: severe obesity, diabe
tes, hypertension, sleep apnea or coronary artery disease. And high percentage will have
metabolic syndrome [31] (see table 7). The prevalence of steatosis and steatohepatitis in
these patients undergoing liver biopsy when surgery is performed, is 91% and 37%, re
spectively [32]. At least a third of morbidity obese patients have portal inflammation, and
this is related to the presence of fibrosis [20, 33]. In early stage, localization of fibrosis dif
fers from those nonbariatric populations, in bariatric is portal and in nonbariatric is perisi
nusoidal [19].
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The Adult Treatment Panel III clinical definition of the metabolic syndrome:
- Requires the presence of three or more of the following features:
Waist circumference greater than 102 cm in men or greater than 88 cm in women.
Triglyceride level 150mg/dl or greater.
High-density lipoprotein (HDL) cholesterol level less than 40 mg/dl in men and less than 50 mg/dl in women.
Systolic blood pressure 130 mmHg or greater or diastolic pressure 85 mmHg or greater.
Fasting plasma glucose level 110 mg/dl or greater.
Table 7. Definition of the metabolic syndrome [31].
NAFLD after bariatric surgery. Improvements of major histological features of disease ac

tivity, grade of steatohepatitis and rarely fibrosis following therapy (dietary, medicines or
surgery) have been reported [34]. After surgical intervention liver histology improve in
these features: lobular steatosis, necroinflammatory changes and fibrosis, against no im
prove in portal abnormalities [35]. Recent meta-analysis [36] shows that patients after bariat
ric surgery have improvement or resolution in steatosis (91,6%), in steatohepatitis (81,3%), in
fibrosis (65,5%) and for complete resolution of NASH was 69,5%.
In the near future, we will have to get used to new types of treatment, for example, meta
bolic surgery, which might be performed to non-morbid obese patients with diabetes, and
to the new changes in hepatic parenchyma following endoscopic procedures performed to
treat obesity.
NAFLD in children. Pediatric NAFLD can have a different histologic presentation than
adult NAFLD. In the first large biopsy series of pediatric NAFLD [37], two different histo
logic patterns were described with differences in race and gender. Type 1 NASH: similar to
adults, more common in Caucasian children. Histologic characteristics are: steatosis, bal
looning degeneration and perisinusoidal fibrosis. On the other hand, Type 2 NASH was
more common in Asian, Native American and Hispanics. Typical features in the liver biopsy
are: steatosis with lymphocytic portal inflammation and portal fibrosis. Children with type 2
were younger and had a greater severity of obesity, and advanced fibrosis. This kind of pat
tern was described in adult morbid obese patients undergoing bariatric surgery, these pa
tients mean age were slightly lower [33]. Overlap cases with characteristic of both
histological types may also be observed in pediatric NASH. A multicentre retrospective co
hort study reviewed 130 liver biopsies of children according to these criteria of pediatric
NAFLD [38]. The majority of the biopsies presented an overlapping pattern (82%). Ad
vanced fibrosis was associated with the presence of lobular and portal inflammation.
Portal fibrosis is common in pediatric NAFLD and may evolve to periportal fibrosis and
bridging fibrosis in some patients, whereas progression to cirrhosis is observed in rare
cases [39].
It is not clear, that Type 2 NASH, described as pediatric NASH, is an entity by itself or it is
another stage of the spectrum of NASH which could be a predictor of those who have a se
vere disease [40].

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8. Imaging tecnology in NAFLD

Ultrasonography (US), computed tomography (CT), and magnetic resonance (MR) can identi
fy liver steatosis but not steatohepatitis, nevertheless they provide anatomical and morpholog
ical information. The sensitivity of these imaging methods is optimal for steatosis over 33%.
When advance liver disease, radiology techniques can provide indirect signs of cirrhosis, such
as portal hypertension, or may be useful for the screening and diagnosis of hepatocellular carci
noma (HCC)[41]. Imaging technique may help to differentiate diffuse from focal form of steato
sis. Hepatic fatty infiltration can present as focal steatosis (a focal area of steatosis in an
otherwise normal liver) or as focal fatty sparing (fatty change with sparing of certain areas) [42].
Abdominal US is the most commonly used imaging technique to clinically evaluate the pres
ence of liver steatosis. Advantages include low cost, lack of radiation exposure and wide availa
bility. The brightness of the liver echo is compared with the kidney, the attenuation of the
sound beam by the fat results in relatively hypoechoic kidney. For detailed description of sono
graphic features for staging fatty liver see table 8. This US feature is not characteristic of NAFLD
because it can be present in other diffuse parenchymal liver disease. The US can be accurate de
tecting hepatic steatosis when there is a moderate to severe infiltration [43]. Overall sensitivity
and specificity are 60-94% and 66-95%, respectively, however the sensitivity is lower when BMI
(body mass index) is over 35 Kg/m2. Although this acceptable level of sensitivity it does not pro
vide reproducible quantitative information. US scoring system for fatty liver is based on hyper
echogenic liver tissue, the increased discrepancy of echo amplitude between liver and kidney
and the loss of echoes from the walls of the portal system [44]. US cannot differentiate between
steatosis and fibrosis, but with advance degrees of fibrosis an increase in coarse echoes without
posterior beam attenuation can be seen.
Features
NORMAL
MILD
MODERATE
SEVERE
Liver echotexture
Liver parenchyma is homogeneous and
Slight increase in echo Intermediate Gross discrepancy of the
no difference in contrast between liver
pattern
increased hepatic to renal cortical
and kidney
echogenicity
Echo penetration an Liver structure is clearly defined from the Mild attenuation of
Intermediate Marked attenuation of sound
visibility of
surface to diaphragm. The outline of the sound beam through
beam through the liver, the
diaphragm
diaphragm is clearly visualized.
diaphragm is not visualized.
the liver
Clarity of liver blood Vessel wall and lumen of vessel can
Slight decrease
vessel structure
definition of portal
visualized with absence of all
venule walls
smaller portal venule walls
clearly visualized
Intermediate Only the main portal walls can be
Table 8. Ultrasonographic grading system for diagnosis of fatty liver, adapted from [41].
Computer Tomography provides an accurate and a reliable visualization of whole liver, so

that not only diffuse but also focal fatty infiltration of the liver parenchyma can be accurately
diagnosed. CT enables the evaluation of absolute measurement of attenuation values which
are given in Hounsfield units, the difference of attenuation between liver and spleen as well as
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the calculation of the liver-to-spleen attenuation ratio, those correlate with steatosis degree.
Liver density as measured by CT attenuation units has been shown to have an inverse correla
tion to the degree of fatty infiltration. Non-enhanced CT provides a high performance in quali
tative diagnosis of hepatic steatosis when fatty infiltration is over 30%, obtaining 82% of
sensitivity and 100% specificity using histologic analysis of biopsies of liver donors as the refer
ence standard [45], however is not sensitive in detecting mild-to-moderate amounts of steato
sis between 5% and 30% [43]. New CT scanning techniques are developing, such as dualsource/dual energy scanners, but their evaluation needs further studies. A drawback of this
technique is the liver iron overload because it increases the attenuation. This method is associ
ated with radiation exposure which limits its use in children.
Magnetic Resonance can detect steatosis by exploiting the difference of resonance frequen
cies between water and fat proton signals. The sensitivity and specificity of MRI in detecting
as low as 5% of liver fat infiltration are 85% and 100%, respectively [46]. The detection of the
fatty liver can be seen in white/bright when applying in-phase T1 images and black
when applying out-of-phase images, compared to the signal intensity of the spleen and par
aespinal muscles. Another technic of MR imaging with fat saturation may quantify more ac
curately liver fat infiltration, especially in patients who have fibrosis.
MR spectroscopy can reliably quantify even minimal steatosis, as low as 0,5% [47]. In has
been based on the ubiquitous protons hydrogen and phosphorus [48], and more than 5% of
fat content on MR spectroscopy indicates presence of steatosis [49]. Its routine application is
limited by cost and lack of availability, and it remains a research tool.
Methods (S/s)
Advantages
Disadvantage
Ultrasonography.
Noninvasive, widely aviable, low cost, repetition,
Operator dependent.
(60-95% / 84-100%)
Useful for screening
Qualitative assessment of steatosis.

Only accurate when moderate-to-severe fat
infiltration.
Computer tomography,
Noninvasive, semiquantitative assessment of fat
Radiation exposure.
Contrast images.
content.
Iron overload, copper and fibrous tissue could be
(50-86% / 75-87%)
Detects focal or diffuse infiltration.
confounding factors.
Not sensitive for mild-to-moderate amounts of
steatosis.
Magnetic resonance imaging
Noninvasive, semiquantitative, and no radiation
Limitation in patients with iron overload.
(85% / 100%)
exposure.
Not suitable for patients with claustrophobia or
Detects focal or diffuse infiltration.
implantable devices.
MR spectroscopy
Noninvasive, reproducible, accurate quantification. High cost not widely available. Long time taking
images.
Table 9. Pros and cons of radiologic modalities for the study of NAFLD. S: sensitivity; s: specificity. Adapted from [41].
US, CT and MR are insensitive in differentiating hepatic steatosis from NASH, and they can
not be used to stage fibrosis [43, 48]. But in the near future, a novel method based on MRI

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imaging and a new software will be able to stage fibrosis and to distinguish NASH from noNASH. Professor Romero-Gomez conducts this study and it will be soon published.
Table 9 summarizes advantages and disadvantages of these radiologic methods.
9. Non-invasive assesment in NAFLD

Liver biopsy remains a useful tool to confirm the diagnosis and exclude other disease or
helps to discover concomitant chronic liver disease. It provides prognostic information by
staging and grading this disease. At present non-invasive diagnostic markers could provide
a new tool for differentiating fatty liver from NASH as well as for grading /staging NAFLD.
The investigation of these new diagnostic methods comes from the well known drawbacks
of liver biopsy. These include sampling error, inadequate biopsy size, variability in patholo
gist interpretation, cost and associated morbidity (complications 0,3%, mortality rate 0,01%).
An ideal non-invasive test should be simple, reproducible, readily available, less expensive
than liver biopsy, able to predict the full spectrum of liver fibrosis stages, and reflect
changes occurring with therapy [48].
Some reviews provide an overview of the role of non-invasive test in NAFLD [48, 50, 51].
We will try to present many of these scores through a table (number 10) to summarize their
characteristics. AUROC is a numerical data that assess the performance of a scoring system.
AUROC value greater than 0,8 indicate good diagnostic performance. The closer the value
to 1, the better performing the scoring system.
SCORE [Reference]
Variables
Cutoff
AUROC
Sens. (%) Spec. (%)
PPV (%)
NPV (%)
6 components of FibroTest-
0,3
0,79-0,86
85
46
93
ActiTest, BMI, cholesterol,
0,7
63
79
68,2
71,4
NONINVASIVE DIAGNOSIS OF STEATOSIS

SteatoTest [52]
88
triglycerides, glucose
Fatty liver Index (FLI) BMI, waist circumference,
<30
[53]
>70
triglycerides, GGT
0,85
87
86
0,763
73,7
65,7
0,83
75
81
NONINVASIVE ASSESSMENT IN NASH

Palekar [54]
HA >55 mcg/l, age >50 years, 3

female gender, AST >45 UI/ml,
AAR >80.
CK-18 [17]
CK-18 plasma (apoptosis

marker)
250 U/l
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SCORE [Reference]
Variables
oxNASH [55]
Detection of lipid peroxidation
Cutoff
AUROC
0,74-0,83
Sens. (%) Spec. (%)
PPV (%)
NPV (%)
63-81
84-97
94
74
94
74
0,73
71,4
72,7
83,3
57,1
0,79
29
98
91
71
56
93
90
85
98,5
44,5
75,4**
75**
products by chromatographymass spectrometric

SHIMADA [56]
Serum adiponectin, type 4

collagen 7s level, HOMA-IR
NASH Diagnostics
CK-18, cleaved CK-18,
[57]*
adiponectin, resistin
NashTest [58]
Age, gender, BMI, triglycerides,
0,3825
cholesterol,
alfa-2macroglobulin, GGT,
haptoglobin, apolipoproteinA1, total bilirubin
NONINVASIVE MANAGEMENT OF FIBROSIS IN NAFLD
NAFLD fibrosis score Age, BMI, IFG/diabetes, AAR,
1,455
[59]
platelet, albumin
0,676
0,88
Pediatric NAFLD
Age, waist circumference and 9: rule in liver 0,85
fibrosis index [60]
triglycerides.
fibrosis.
<3:rule out
fibrosis
**Pre-test probability:
69%
BARD score [61]
FIB-4 [62]
BMI 28 =1, AAR 0,8 =2,
2: advanced
diabetes=1,
fibrosis
Age, AST, platelet, ALT
<1,3
0,81
96
0,80
43
90
80
83
86
54
93
90
71
94
70
80
70
80
No fibrosis 0,76
61
80
81
79
0,84
100
47
F0-1 VS F2-4:
70
>2,67
APRI [63]
AST, platelet
0,98
Advanced fibrosis: 75
0,85
ELF [64]
HA, TIMP1, P3NP.
0,3576
For severe fibrosis 80

0,90.
-0,1068
For moderate
fibrosis 0,82
ELF: -0,2070.
BAAT [65]
Age 50years, BMI 28kg/m2 0-1, for septal
100
=1, triglycerides 7mmol/L=1, fibrosis

ALT 2N
Fibrotest [66]
0,30
075-085.
90

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SCORE [Reference]
Variables
Cutoff
AUROC
Sens. (%) Spec. (%)
PPV (%)
NPV (%)
F0-2 VS F3-4:
0,81-0,92
0,70
Fibrometer [67]
Glucose, AST, platelets, ferritin, <0,611
F0-1 vs F2-4:
ALT, weight, age.
0,936-0,952
98
73
ULTRASOUND BASED TECHNIQUES IN FIBROSIS DETECTION IN NAFLD

Fibroscan [68]
Transient elastography
9,9 kPa
F 3: 0,99
100
93
77
100
16 kPa
F4: 0,998
100
93
86
100
ARFI [69]
4,24 kPa
F0-2 vsF3-4: 0,90 90
90
ARFI [68]
1,77 m/sec
F 3: 0,973
100
91
71
100
1,90 m/sec
F4: 0,976
100
96
75
100
SS vs NASH: 0,93
94
73
85
89
MAGNETIC RESONANCE IMAGING BASED TECHNIQUES

MR elastography[70] Discriminating NASH from 2,74 kPa
simple steatosis (SS)
Means:
Simple steatosis.
- 2,51kPa.
NASH no fibrosis.
- 3,24kPa.
With fibrosis
- 4,16kPa.
Abbreviations: AUROC: area under receiver operator curve. Sens.: Sensitivity; Spec.: Specificity; PPV: positive predictive
value; NPV: negative predictive value; BMI: body mass index; GGT: Gamma-glutamyl-transpeptidase; HA: hyaluronic
acid; AST: aspartate transaminase; AAR: AST/alanine aminotransferase, TIMP-1: tissue inhibitor of matrix metallopro
teinase 1; P3NP: aminoterminal peptide of pro-collagen III; ARFI: acoustic radiation force impulse; * Sample: bariatric
surgery patients. COMMENTS: BARD score can reliably exclude advance fibrosis, particularly among non-diabetics.
FIB-4, as happened with BARD score, is useful in excluding advance fibrosis due its high NPV. Transient elastography
and ARFI are based on the variation of the speed wave through liver tissue (generated by vibrator/short-duration
acoustic pulses, respectively), this can be measured and converted to a numerical value (in kPa and m/sec, respectively,
but ARFI could also be expressed as kPa) which is the liver stiffness and it is proportional to liver fibrosis. An important
difference between both systems is that ARFI consists in a probe which can be plugged to a common US machine so
both techniques can be performed at the same time.
Table 10. Non-invasive assessment of NAFLD.
APRI and FIB-4 have been evaluated in obese children and they might be useful in this special
population [71]. Pediatric NAFLD scores is a noninvasive model evaluated in obese children,
and it may help clinicians to predict liver fibrosis but external validation is needed [60].
In the future, new serologic markers, such as CD36, will help to differentiate more accurate
ly between NAFLD stages, we would be able to distinguish simple steatosis from NASH.
Although clinical and laboratory models may be useful in identifying a group of patients at
a low risk of advance fibrosis and liver biopsy might be avoided, they are not enough for
staging and prognostic purposes if patients are at risk of advance fibrosis [48]. NAFLD Prac
tice Guideline of 2012 recommends NAFLD fibrosis score to identify patients with higher
likelihood of having bridging fibrosis and/or cirrhosis [1].
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10. New diagnostic platforms in nafld

NAFLD is a disease with wide spectrum: from steatosis through inflammation to fibrosis
and finally cirrhosis and hepatocellular carcinoma, even in absence of cirrhosis [72]. The
strongest predictor of fibrosis progression in NAFLD is steatohepatitis. The most important
features are hepatocellular degeneration (ballooning) and inflammatory cell infiltration.
These new techniquesinclude genomics, metabolomics and proteomics.
Genomics. Gene expression studies provide an insight into possible mechanism of patho
genesis as well as potential biomarkers of disease. One method for studying gene expression
is micro-arrays of DNA. A study using this test found 34 gens with different expression in
NASH vs controls, these genes where implicated in lipid metabolism and extracellular ma
trix remodelling [73]. Another study compared gene expression in NASH-related cirrhosis
with other causes of cirrhosis. In NASH cirrhosis group genes involved in anti-oxidant
stress were underexpressed, along with genes involved in fatty and glucose metabolism
[74]. In our centre we used micro-arrays to study gene expression in obese patients with
NAFLD [75]. Obese patients with NASH without fibrosis show an overexpression of proin
flammatory and proapoptotic genes; and those with fibrosis show an overexpression of fi
brogenic genes, including the leptin receptor Ob-Rb.
Most recent genomic tests, such GWAS (Genome-wide association studies) provide a method
for evaluating a large number of single nucleotide polymorphisms (SNP) with the same experi
ment. A study performed with a GWAS study found a SNP in farnesyldiphosfatasefarnesyl
transferase 1 (FDFT1) which was associated with different histological parameters (a SNP with
portal inflammation and another different SNP with fibrosis stage) and the total NAFLD activ
ity score [76]. In an earlier GWAS study [77], an SNP in PNPA3 (adiponutrin/patatin-like phos
pholipase-3) was strongly associated with both hepatic fat content and hepatic inflammation.
The prevalence of this mutation may explain the difference in susceptibility to NAFLD seen in
different ethnicities [77]. A subsequent study [78], confirmed the relationship between this
SNP and histological score, the no association with metabolic syndrome.
These studies are incredibly interesting and they could help the development of new nonin
vasive markers, nevertheless all of them share limitations, mainly concerning to sample size.
It is easily understandable given the fact that they use expensive and complex tools [51].
Proteomics. Proteomic tools look specifically at protein expression patterns and profiles.
There are several approaches to proteomic studies depending on the used tool. These tools
are complex; they are based on diverse types of mass-spectrometry. For more detailed infor
mation refer to [79, 80]. The different proteomic platforms support the use of either liver tis
sue or blood. This platform allows identifying, quantifying and comparing proteins in the
study groups of interest. That novel approach has been applied for the study of NAFLD
[81-84]. These studies have found several proteins related to disease progression: alfa and
beta-hemoglobin [84], lumican and FABP1 (fatty acid binding protein-1) [82]; and finally fi
brinogen beta-chain, retinol binding protein-4, serum amiloyd p-component, lumican, trans
grelin-2 and CD5-like antigen, in 6-panel model and complement component 7, transgrelin-2

http://dx.doi.org/10.5772/52622
and insulin grow factor acid labile subunit, in a 3-panel model. These panels performed in
the diagnosis of the diverse NAFLD stages get an area under the receiver operator curve
(AUROC) ranging from 0,83 to 0,91 [83].
Metabolomics. In the natural history of NAFLD the progression to hepatic fibrosis occurs
only in 10 to 25% of cases, leading to cirrhosis, end-stage liver disease or hepatocellular car
cinoma. The strongest predictor of fibrotic progression, apart from pre-existing fibrosis, is
steatohepatisis. A two-hit model has been proposed as an explanation for why some pa
tients progress to NASH. In a first step, because of insulin resistance, adipose tissue has en
hanced triglyceride lipolysis, which leads to increased serum free fatty acids, and impaired
hepatic triglyceride export. In this model, hepaticsteatosis (hit 1) exposes the liver parenchy
ma to environmental and extracellular hepatic insults (hit 2), leading to inflammation, stea
tonecrosis and fibrosis. Impaired mitochondrial oxidation and lipid export may also
contribute to hepatic fat deposition.
Leptin system is also implicated, and its receptor expression is related to fibrosis degree [85].
As it was explained in the introduction, irisin is a newly identified hormone. Irisin is pro
duced in muscle cells induced in exercise [86-88]. Irisin activates changes in adipose tissue,
and make its change from white adipose tissue to brown adipose tissue, and this causes a
significant increase in total body energy expenditure and resistance to obesity-linked insulin
resistance. So this advance opens new pathogenic pathways in NAFLD.
Inflammation is considered to be the central clue for the progression of NAFLD, the origins
and components are considered in this review [89]. Hepatocytes injured by toxic lipid mole
cules play a central role in the recruitment of innate immunity involving Toll-like receptors
(TLR), Kuppfer cells, lymphocytes and neutrophils and possibly inflammasome. On this
way, a study was carried to determine the lipidomic signature in NAFLD [90]. Using proteo
mic tools (mass spectrometry) the investigators found metabolites from nonenzymatic oxi
dation product of arachidonic acid and from impaired peroxisomal polyunsaturated fatty
acid (PUFA). This study links to another, where investigators characterize metabolic profile
to distinguish steatosis and NASH [91], they also found arachidonic acid, among other sub
stances, relation to NASH and fibrosis. Metabolomics analysis was performed to NAFLD
patients showing a lower concentrations of glutathione, an antioxidant substance, in this
group [92].
The key pro-inflammatory signalling pathways in NASH are nuclear factor-kappa B (NFkB) and c-Jun N-terminal kinase (JNK). It could be possible that inflammation in NASH
could originate outside the liver. Gut microbiota, the related Kupffer/TLR response, in
flamed adipose tissue and circulating inflammatory cell can contribute or act as co-factors
that triggers or maintain hepatic injury. In a study conducted in our centre to study the rela
tionship between endotoxemia and NAFLD, we found higher levels of LBP (Lipopolysac
charide-binding protein) in patients with NASH when compared to patients with simple
steatosis [93]. The LBP increase correlates with the level of tumor necrosis factor alfa (TNFalfa) which is overexpressed in patient with NASH and significant fibrosis. [94] Detailed in
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180
formation in pathophysiology of NAFLD and NASH is not the aim of this paper, if you are
interested refer to this review [89].
11. Conclusion
NAFLD is an emerging problem. The study of pathology is ever evolving which is allowing
the development of new therapeutic targets, and the emergence of new diagnostic techni
ques allow better identification of patients who will benefit from new treatments.
Author details
Joaqun Cabezas1, Marta Mayorga2 and Javier Crespo1
1 Gastroenterology and Hepatology Unit, University Hospital Marqus de Valdecilla,
Santander, Spain
2 Pathology Department, University Hospital Marqus de Valdecilla, Santander, Spain
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tologic spectrum of nonalcoholic fatty liver disease. Hepatology. 2009;49(4):1375-84.
Epub 2009/03/31.
[83] Bell LN, Theodorakis JL, Vuppalanchi R, Saxena R, Bemis KG, Wang M, et al. Serum
proteomics and biomarker discovery across the spectrum of nonalcoholic fatty liver
disease. Hepatology. 2010;51(1):111-20. Epub 2009/11/04.
[84] Trak-Smayra V, Dargere D, Noun R, Albuquerque M, Yaghi C, Gannage-Yared MH,
et al. Serum proteomic profiling of obese patients: correlation with liver pathology
and evolution after bariatric surgery. Gut. 2009;58(6):825-32. Epub 2008/04/12.
[85] Cayon A, Crespo J, Mayorga M, Guerra A, Pons-Romero F. Increased expression of
Ob-Rb and its relationship with the overexpression of TGF-beta1 and the stage of fib
rosis in patients with nonalcoholic steatohepatitis. Liver Int. 2006;26(9):1065-71. Epub
2006/10/13.
[86] Kelly DP. Medicine. Irisin, light my fire. Science. 2012;336(6077):42-3. Epub
2012/04/12.
[87] Pedersen BK. A muscular twist on the fate of fat. N Engl J Med. 2012;366(16):1544-5.
Epub 2012/04/20.
[88] Bostrom P, Wu J, Jedrychowski MP, Korde A, Ye L, Lo JC, et al. A PGC1-alpha-de
pendent myokine that drives brown-fat-like development of white fat and thermo
genesis. Nature. 2012;481(7382):463-8. Epub 2012/01/13.
[89] Farrell GC, van Rooyen D, Gan L, Chitturi S. NASH is an Inflammatory Disorder:
Pathogenic, Prognostic and Therapeutic Implications. Gut Liver. 2012;6(2):149-71.
Epub 2012/05/10.
[90] Puri P, Wiest MM, Cheung O, Mirshahi F, Sargeant C, Min HK, et al. The plasma lipi
domic signature of nonalcoholic steatohepatitis. Hepatology. 2009;50(6):1827-38.
Epub 2009/11/26.
[91] Barr J, Vazquez-Chantada M, Alonso C, Perez-Cormenzana M, Mayo R, Galan A, et
al. Liquid chromatography-mass spectrometry-based parallel metabolic profiling of
human and mouse model serum reveals putative biomarkers associated with the
progression of nonalcoholic fatty liver disease. J Proteome Res. 2010;9(9):4501-12.
Epub 2010/08/06.
[92] Kalhan SC, Guo L, Edmison J, Dasarathy S, McCullough AJ, Hanson RW, et al. Plas
ma metabolomic profile in nonalcoholic fatty liver disease. Metabolism. 2011;60(3):
404-13. Epub 2010/04/29.
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[93] Ruiz AG, Casafont F, Crespo J, Cayon A, Mayorga M, Estebanez A, et al. Lipopoly
saccharide-binding protein plasma levels and liver TNF-alpha gene expression in
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non-alcoholic steatohepatitis. Obes Surg. 2007;17(10):1374-80. Epub 2007/11/15.
[94] Crespo J, Cayon A, Fernandez-Gil P, Hernandez-Guerra M, Mayorga M, DominguezDiez A, et al. Gene expression of tumor necrosis factor alpha and TNF-receptors, p55
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Epub 2001/12/04.
Chapter 9
Liver Biopsy After Liver Transplantation

Alpna R. Limaye, Lisa R. Dixon and Roberto J. Firpi
http://dx.doi.org/10.5772/52617
1. Introduction
Histological evaluation of liver allograft biopsies is an integral part of the management of
liver transplant patients. From the time of donor hepatectomy onward, the allograft is sus
ceptible to multiple insults, including warm and cold ischemia, complications related to sur
gical anastomoses, acute cellular rejection, and recurrence of underlying liver disease. It is
often quite challenging to distinguish these various entities by their clinical presentation
alone. In these situations, evaluation of a liver biopsy is frequently necessary to confirm the
diagnosis, to stage recurrent fibrosis, or to monitor response to treatment.
2. Post-transplant liver biopsy techniques

Liver biopsies can be performed with various techniques, including a percutaneous ap
proach (with marking by percussion/palpation, marking by ultrasound (US), or under realtime US or computed tomography (CT) guidance), a transjugular approach, or a surgical/
laparoscopic approach. Although percutaneous liver biopsies on non-transplant patients can
be done without the use of imaging, it is recommended that patients who have undergone
any abdominal surgery (including liver transplantation) undergo biopsies aided by the use
of US to avoid vascular or other structures [1]. While US marking followed by biopsy is suf
ficient in most post-transplant patients, in certain situations (such as split-liver recipients),
biopsy under real-time US or CT guidance is preferred to avoid encountering intervening
bowel loops. While specimens at least 1.5cm in length and containing at least 6-8 portal tri
ads are considered adequate for the diagnosis of chronic liver disease [2,3], some advocate a
minimum length of 2.0 cm and at least 11 complete portal tracts for accurate grading and
staging of liver disease [4].
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Percutaneous liver biopsy can be performed rapidly and safely in an outpatient setting with
the appropriate monitoring equipment and staff availability [5]. After discharge, patients are
typically instructed to avoid strenuous physical activity or driving for 24-48 hours, and are
asked to contact the clinical provider in the event of concerning symptoms. In our institu
tion, a review of over 3,000 liver biopsies (including liver transplant patients) demonstrated
that the majority of complications were discovered within the first hour after percutaneous
liver biopsy, and that shortening the recovery time to 1-2 hours did not impact the frequen
cy of complications [6].
Percutaneous liver biopsy can be performed with suction needles (such as Jamshidi needle
or Menghini needle), cutting needles (such as the Tru-Cut needle), or spring-loaded needle
guns. Specimens adequate for diagnosis, grading, and staging can usually be obtained by
all of the biopsy needles used in current practice.
In patients with severe/uncorrectable coagulopathy, thrombocytopenia (typically platelet
count < 50,000/mm3), large ascites, morbid obesity, or an inability to cooperate, a transjugu
lar liver biopsy (TJLB) is typically recommended [7]. In addition, TJLB is useful in patients
for whom wedged hepatic venous pressure gradient (HVPG) measurement would be clini
cally useful. Miraglia et al reported on the safety of TJLB in liver transplant patients, with
only one complication in 183 biopsies (0.5%) [8].
TJLB is typically performed with the use of automated needle systems, such as the
Quick-Core needle and the Flexcore needle. It has been established that these automated
needle systems often require multiple passes, and usually collect smaller core samples
than those obtained by percutaneous liver biopsy [9]. Despite this fact, specimens ob
tained via TJLB are adequate for diagnosis, staging, and grading liver disease in greater
than 90% of cases [10,11].
Surgical liver biopsies (either open liver biopsy or laparoscopic liver biopsy) are typically
performed when patients require a surgical procedure for another indication. In liver trans
plant patients, this often involves repair of postoperative hernias. Biopsies in this setting can
be performed with either automated needle systems or with a wedge resection, and the pro
cedure provides the advantage of direct visualization of the liver and the ability to immedi
ately diagnose and treat any bleeding which occurs.
3. Complications of liver biopsy

Although invasive, liver biopsy is a relative safe procedure, whether performed percutane
ously or via the transvenous route. In a review of over 60,000 non-transplant patients, death
within seven days directly related to liver biopsy occurred in 1 out of every 10,000 proce
dures, and all-cause mortality within seven days occurred in approximately 0.2% of patients
[12]. Serious complications were similarly rare, with pain occurring in 2% of patients, hemo
peritoneum occurring in 0.04%, and hemobilia occurring in 0.01% [12]. Similarly, studies of
allograft liver biopsies demonstrate a mortality rate of up to 0.2%, and a rate of major com

http://dx.doi.org/10.5772/52617
plications between 0.2% and 1.8% [13]. While early studies suggested an increased risk of
post-biopsy sepsis in patients with Roux-en-Y choledochojejunostomy, subsequent studies
show that the risk is similar to patients with a duct-to-duct anastomosis [14].
4. Post-transplant liver enzyme abnormalities

Abnormalities in liver enzyme levels are often encountered in liver transplant patients, and
can represent hepatocellular injury (reflected by the transaminases), biliary injury [reflected
by alkaline phosphatase or gamma-glutamyl transferase (GGT)], or hepatic synthetic dys
function (reflected by the albumin or by coagulation abnormalities). While the use of serum
blood tests (such as viral or autoimmune serologies) and imaging techniques (such as ultra
sound with Doppler, angiography, and magnetic resonance cholangiography) can be useful
to determine the etiology of abnormal liver enzymes, liver biopsy is often necessary for a
definitive diagnosis.
5. Early post-transplant liver enzyme abnormalities

The differential diagnosis of liver enzyme abnormalities varies with the amount of time
which has passed since liver transplant. In the normal post-transplant course, liver enzymes
typically rise immediately following transplant and become normal or near-normal within
3-5 days. If the enzymes fail to improve or normalize but soon rise again, it is likely that an
early complication has occurred (Table 1). In the very early post-transplant period (within
the first week), liver enzyme abnormalities can be related to primary graft nonfunction
(PNF) or dysfunction, hepatic arterial insufficiency, small for size syndrome (SFSS), or portal
venous thrombosis (PVT).
5.1. Primary graft dysfunction
PNF and primary graft dysfunction are associated with prolonged ischemia time [15], and it
is likely that preservation and reperfusion injury play a role. PNF is heralded by a precipi
tous rise in hepatic transaminases in the second or third postoperative day accompanied by
signs of hepatic failure (encephalopathy and coagulopathy). Once hepatic artery thrombosis
(HAT) has been ruled out by imaging, a liver biopsy confirms the diagnosis. Biopsies in this
setting typically show centrilobular hepatocyte dropout due to hepatocellular necrosis, with
compensatory zone 2 hepatocyte proliferation and bile ductular proliferation [16].
5.2. Hepatic artery thrombosis
The clinical presentation of HAT is quite similar to that of PNF, with a dramatic increase in
hepatic transaminases and bilirubin in the very early post-transplant period. A liver biopsy
is typically not required, as the diagnosis can usually be confirmed with angiography. When
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performed, biopsies of patients with hepatic arterial insufficiency show hepatocyte foamy
degeneration or necrosis and features of ischemic cholangitis [17].
Disease/
Incidence
Complication
Time of
Clinical presentation
Risk Factors
presentation
Histological
Characteristics
Preservation/
Up to 30%
Reperfusion
(2-7% severe)
Within 3 days Elevated transaminases, Prolonged cold or warm

bilirubin, INR.
Injury
[15]
Encephalopathy in
ischemia time, greater
Centrilobular
hepatocyte
than 30% donor steatosis dropout, zone 2
severe injury
hepatocyte
proliferation, bile
duct proliferation
[16]
Hepatic artery
3-10% in
Day 2 to 7 post-
Severe elevation of
Technical/anastomotic
Foamy
thrombosis
adult
transplant
transaminases, bilirubin,
complications
hepatocyte
transplant (up
alkaline
degeneration,
to 40% in
phosphatase/GGT
features of
pediatric
ischemic
transplant)
cholangitis
[17]
Acute Cellular
24-80% by 6
Typically 2-3
Rejection
months post-
weeks after
transplant
[26]
Elevated transaminases, Younger recipient, older

bilirubin, alkaline
transplant, up to phosphatase. Possible

3 months posttransplant
donor, history of
Portal
inflammation,
autoimmune disorder, ?
biliary
recent history of
female recipient
inflammation,
inadequate
[17]
endothelitis
Often normal,
immunosuppression
Portal vein
Less than 1% Early: within first
thrombosis
Early: acute hepatic
Hypercoagulable state,
week post-
failure
prior history of PVT
transplant
Late: ascites, variceal
Late: within first
bleeding
nodular
year post-
hyperplasia [21,
transplant
22]
Small for size
Not well
Sequelae of
Ascites, spontaneous
syndrome
defined, but
portal
bacterial peritonitis,
greatest when hypertension: 6

graft-to-
may show
features of focal
Graft-to-recipient body
Centrilobular
weight ratio less than 0.6 cholestasis and
variceal bleeding
to 12 months
recipient body post-transplant

weight ratio is
less than 0.6
[18]
Table 1. Differential diagnosis of early post-transplant liver enzyme abnormalities
[18, 19]
steatosis,
interface bile duct
inflammation [20]

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5.3. Portal vein thrombosis

Early acute PVT presents clinically as acute hepatic failure and demonstrates histological
features of hepatocyte necrosis. Occasionally, PVT presents later, in which case the features
of portal hypertension dominate. In this situation, if a biopsy is performed, it can be normal
or show features of nodular regenerative hyperplasia [18,19].
5.4. Small for size syndrome
Patients with SFSS present with dominant features of portal hypertension, such as ascites
and variceal bleeding. This syndrome is the result of relative portal hyperperfusion com
pared to hepatic arterial blood flow, and often occurs when the transplanted liver (or liver
segment) is less than 0.6- 0.8% of the recipient body weight [20,21]. The diagnosis is typically
made clinically, with signs of portal hypertension such as ascites and variceal bleeding. If a
liver biopsy is performed, the allograft typically shows centrilobular cholestasis, centrilobu
lar steatosis, and interface ductular proliferation [22].
5.5. Acute cellular rejection
The most common cause of early allograft dysfunction is acute cellular rejection (ACR).
Although most cases of ACR occur in the first three months post-transplant (most often
in the second or third post-transplant week), late-onset ACR (occurring up to 10 years
post-transplant) has been reported [23]. ACR typically presents as moderate to severe el
evations in hepatic transaminases and alkaline phosphatase/GGT, with some degree of
bilirubin elevation, often in patients with a recent history of inadequate immunosuppres
sion. The gold standard for the diagnosis of ACR remains the liver biopsy, which typi
cally shows variable degrees of mixed portal inflammation (often with increased
eosinophils), bile duct inflammation, and endotheliitis (typically in the portal vein or cen
tral vein branches) (Figure 1). The Banff criteria are the most widely used to describe
ACR, and give a score of 1-3 for each component. These scores are added to calculate
the Rejection Activity Index (RAI), which ranges from 3-4 (mild ACR) up to greater than
7 (severe ACR) [24]. It should be noted that late-onset ACR often appears quite different
histologically, with a greater likelihood of lobular inflammation, less prominent bile duct
inflammation, and a tendency towards monotypic portal inflammation [25]. A follow-up
liver biopsy after 3-7 days of increased immunosuppression is occasionally used to con
firm response to treatment.
5.6. Late post-transplant liver enzyme abnormalities
Beyond three months post-transplant, the differential of liver enzyme abnormalities
changes. Broadly, the diagnoses can be categorized as chronic rejection, native disease recur
rence, de novo infectious complications, toxic complications, de novo hepatitis, or vascular
complications (Table 2).
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Figure 1. Acute rejection, with endotheliitis (arrowhead), bile duct destruction (arrow), and mixed portal inflamma
tion (40x, H&E)

http://dx.doi.org/10.5772/52617
Disease/
Incidence
Complication
Chronic rejection
Time of
Clinical presentation
Risk Factors
presentation
3-5%
3-12 months after

transplant
Histological
Characteristics
Rising alkaline
Inadequate
Bile duct atrophy/loss,
phosphatase/GGT, late immunosuppression, history foamy arteriopathy [26]

elevation in bilirubin
of multiple episodes of or
ongoing acute cellular
rejection [17]
Recurrent HCV
Near-universal
Re-infection
Elevated transaminases
within 72 hours; in typical recurrent HCV
FCH: excessive
Portal inflammation,
immunosuppression
interface hepatitis, lobular
histological
FCH: jaundice, marked
activity [28-30]
recurrence within
elevation of alkaline
FCH: cholestasis, fibrosis
1-2 weeks;
phosphatase/GGT,
[35]
clinically
extremely high HCV viral
significant
load
recurrence within
3 years (within 1
year for FCH) [27]
Recurrent HBV
Less than 10%
Typical recurrent Elevated transaminases,
Inadequate prophylaxis
with adequate HBV: 6-12 months elevated HBV viral load
Lymphoplasmacytic portal
inflammation, Kupffer cell
prophylaxis [36] post-transplant
hypertrophy, lobular
FCH: within 1
disarray; ground-glass
month post-
hepatocytes; positive
transplant
immunostaining for
hepatitis B surface antigen
and core antigen [37]
Recurrent AIH
Up to 40% [38]
Variable
Slow progression of
Inadequate
transaminase elevation immunosuppression, native

type II AIH
CMV infection
5-8% with
1-12 months
prophylaxis
post-transplant
CMV hepatitis: Elevated Graft from CMV-antibodytransaminases

Extrahepatic CMV:
positive donor into CMV-
Lymphoplasmacytic portal
infiltrate, prominent
interface activity [29]
Portal inflammation,
hepatocytes with CMV
antibody-negative recipient inclusions, focal bile duct
gastroenteritis, colitis,
damage [29]
pneumonitis
EBV infection
Up to 80% of
6-12 or more
EBV hepatitis: usually
patients who are
months post-
asymptomatic
EBV-antibody-
transplant
PTLD: lymphoma-like
Progression to PTLD:
presentation
excessive
+EBER
immunosuppression,
PTLD: immunoblasts, with
negative at time
of transplant
Primary infection: EBV-
EBV hepatitis: portal and
antibody-negative recipient sinusoidal infiltrates with

atypical lymphocytes,
preceding CMV infection
varying degrees of
[43]
architectural distortion [45]
Table 2. Differential diagnosis of late post-transplant liver enzyme abnormalities
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5.7. Chronic rejection

Chronic rejection involves immune-mediated injury to the hepatic arterial endothelium and
bile duct epithelium. It is most commonly seen in patients who have experienced repeated
bouts of significant ACR and/or have a recent history of inadequate immunosuppression.
The typical clinical presentation is a slow rise in alkaline phosphatase/GGT, often followed
by a rise in bilirubin. Procurement of an adequate biopsy sample (with at least ten complete
portal triads) is crucial in the histologic diagnosis of chronic rejection [26]. The minimal cri
teria for diagnosis of chronic rejection, as defined by the 2000 Banff recommendations, are
(1) bile duct atrophy/pyknosis affecting a majority of bile ducts (with or without bile duct
loss); (2) foam cell obliterative arteriopathy (Figure 2); or (3) bile duct loss in greater than
half of the portal tracts [26].
Figure 2. Foamy arteriopathy in the setting of chronic rejection (H&E 10x)
In the months and early years following liver transplant, recurrence of the underlying dis
ease which led to transplant becomes a common problem. Disease recurrence can be viral
(most commonly hepatitis B or C), immunological (such as autoimmune hepatitis, primary
sclerosing cholangitis [PSC], or primary biliary cirrhosis [PBC]), metabolic (such as non-alco
holic fatty liver disease [NAFLD]), malignant (hepatocellular carcinoma or cholangiocarci
noma), or idiopathic. The diagnosis of recurrent PSC, PBC, NAFLD, and malignancy is
relatively straightforward and is therefore not discussed further. However, the degree of
clinical and histological overlap between entities such as rejection, recurrent viral hepatitis,
and autoimmune hepatitis can create diagnostic conundrums without close clinicopatholog
ical correlation.

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5.8. Recurrent hepatitis C

Recurrent hepatitis C (HCV) infection is a universal phenomenon after liver transplantation,
and exhibits an accelerated progression to advanced liver disease [27]. Particularly in the
early months after transplant, the differential diagnosis of abnormal liver enzymes in HCV
patients includes both ACR and recurrent HCV. These entities are usually distinguished his
tologically. Histologically established recurrent HCV demonstrates portal inflammation, of
ten with lymphoid aggregates, interface hepatitis, and lobular disarray [28-30] (Figure 3).
There is often a component of ductular reaction, which is uncommon in HCV in native liv
ers [30]. While endotheliitis was traditionally considered specific to rejection, recent data
demonstrate portal branch endothelitis in biopsies of native HCV livers [31,32]. It does ap
pear that moderate to severe central vein branch endotheliitis remains fairly specific for re
jection. In a prospective analysis of biopsies from 48 HCV transplant patients, Demetris et al
described strict criteria to avoid overdiagnosis of ACR: (1) inflammatory bile duct injury in
at least 50% of portal tracts, and/or (2) mononuclear perivenular inflammation with hepato
cyte necrosis in at least 50% of terminal hepatic venules [33]. In cases where the differentia
tion of ACR and recurrent HCV is not clear, the use of an immune function assay can be a
useful adjunct [34]. Occasionally, recurrent HCV presents aggressively, in an entity known
as fibrosing cholestatic hepatitis (FCH). The risk of FCH increases with aggressive immuno
suppression, such as that used to treat ACR. Histologically, FCH is distinguished by a prom
inent component of cholestasis and fibrosis [35].
Figure 3. Recurrent HCV, with chronic portal inflammation (ellipse), and interface as well as lobular activity (H&E,10x)
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5.9. Recurrent hepatitis B

Recurrent hepatitis B (HBV) infection was common in the era before combination prophy
laxis with hepatitis B immunoglobulin and oral antiviral agents. The current rate of recur
rent HBV (less than 10%) is attributed to a lack of prophylaxis for various reasons [36].
Histologically, recurrent HBV demonstrates lymphoplasmacytic portal inflammation,
Kupffer cell hypertrophy, and lobular disarray [37]. Ground glass cells containing HBV sur
face antigen are often seen. Immunostaining demonstrates HBV surface antigen in hepato
cyte cytoplasm and HBV core antigen in hepatocyte nuclei. Recurrent HBV can also cause
FCH, characterized by cholestasis, perisinusoidal fibrosis, and swollen hepatocytes with im
munoreactivity for HBV core antigen [37]. In patients without demonstrable HBV core anti
gen staining, other causes of hepatic dysfunction should be sought.
5.10. Recurrent and de novo autoimmune hepatitis
Recurrence of autoimmune hepatitis (AIH) can occur in up to 40% of patients, but the course
is typically slowly progressive [38]. The biochemical/serological diagnosis of AIH [39] can be
difficult in post-transplant patients, and therefore a liver biopsy is often required for a defin
itive diagnosis. In the chronic phase, recurrent AIH demonstrates lymphoplasmacytic portal
infiltrate with prominent interface activity, perivenular activity and variable degrees of lob
ular necroinflammatory activity [29] (Figure 4). In patients without a pre-transplant history
of AIH, the findings of lymphoplasmacytic infiltrate and perivenular activity, the differen
tial diagnosis includes recurrent HCV, rejection, and de novo AIH. This distinction relies on
close clinicopathological correlation which takes into account the timing of onset, the immu
nosuppressive state, and the degree of perivenular damage [40].
Figure 4. Portal area with interface hepatitis, numerous plasma cells (arrowhead) and scattered eosinophils (arrow)
(H&E, 40x)

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Liver transplant patients are also at risk of de novo infections due to their immunosup
pressed state. While the diagnosis of most of these infections is fairly straightforward, posttransplant cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infection can be more
difficult.
5.11. CMV infection
CMV is the most common clinically significant viral infection after solid organ transplanta
tion, with an incidence of up to 30% prior to the use of routine prophylaxis [41]. The risk of
post-transplant CMV infection is greatest in CMV-antibody-negative recipients who receive
a graft from a CMV-antibody-positive donor. Clinically, CMV infection can present with fe
ver, myelosuppression, and/or organ involvement (such as gastritis, colitis, hepatitis, or
pneumonitis). While detection of CMV in the serum can provide a rapid diagnosis, a liver
biopsy is often required to distinguish CMV from allograft rejection or demonstrate that
both entities are present [42]. Typically, CMV hepatitis is characterized by mononuclear or
mixed portal inflammation, focal bile duct damage, and hepatocytes with CMV inclusions
(large eosinophilic nuclear inclusions surrounded by a clear halo [29] (Figure 5). Although
some features similar to allograft rejection (portal lymphocytic inflammation, mild endothe
liitis) can be seen in CMV hepatitis, immunostaining for CMV antigens and/or the presence
of CMV inclusions confirms that CMV is the driving force behind the hepatic dysfunction.
Figure 5. Hepatocyte with intranuclear CMV inclusion (arrowhead) (H&E, 100x)
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5.12. EBV infection and post-transplant lymphoproliferative disorder

The clinical presentation of EBV infection can vary from asymptomatic hepatitis to posttransplant lymphoproliferative disorder (PTLD). Patients without pre-transplant immunity
to EBV are at the greatest risk of infection. Patients with primary post-transplant EBV infec
tion, those with previous symptomatic CMV infection, and those with recent excessive im
munosuppression are at the highest risk for progression to PTLD [43]. EBV hepatitis
typically demonstrates portal and sinusoidal infiltrates consisting of atypical lymphocytes.
Often the lymphocytes are arranged in a single-file pattern within sinusoids [29]. Another
histological pattern which can be seen in EBV hepatitis consists of mixed periportal and si
nusoidal infiltrates with large atypical mononuclear cells and immunoblasts, mild bile duct
damage, and hepatic lobular activity [44]. The finding of EBV-encoded RNAs (EBERs) is
confirmatory in most cases.
PTLD is a heterogeneous lymphoproliferative disease divided into three main categories:
early lesions, polymorphic PTLD, and monomorphic PTLD [45]. Early lesions demonstrate
plasmacytic hyperplasia, and may or may not have prominent immunoblasts [29,45]. Poly
morphic PTLD is characterized by mixed infiltrates of monoclonal or polyclonal plasma
cells, immunoblasts, and destruction of the underlying lymphoid architecture [45]. In
monomorphic PTLD, most cases arise from B cell populations which demonstrate inva
sion, architectural effacement, and cellular atypia [29]. A fourth category, Hodgkins lym
phoma-like PTLD, is sometimes described [46], and appears histologically like Hodgkins
lymphoma which occurs in non-transplant patients. PTLD patients with positive EBER re
sults may represent relatively better histopathological features than patients with EBERnegative PTLD [47].
6. Indication and protocol liver biopsies

The majority of post-transplant liver biopsies are performed in response to changes in liver
enzyme levels and/or abnormal imaging findings. Particularly in the early post-transplant
period, these so-called indication biopsies are usually diagnostic and often result in a
change in management. However, as patient and graft survival continues to improve, it has
become clear that normal histology is rarely seen in the long-term liver graft [48]. What is
not clear, however, is whether the histologic abnormalities seen in most late allograft biop
sies correlate to clinically-significant disease, and whether the routine use of so-called pro
tocol liver biopsies (performed at regular time points despite normal liver enzyme levels) is
clinically justified. In our institution, we no longer perform annual protocol biopsies on pa
tients with alcoholic liver disease, non-alcoholic fatty liver disease, or cryptogenic liver dis
ease. The use of annual or semi-annual protocol biopsies in patients with AIH, PSC, or PBC
is left to the discretion of the treating provider and/or the desires of the patient. In all HCV
patients, protocol biopsies are performed at four months post-transplant, at one year posttransplant, and annually thereafter for at least the first five years. In contrast, an informal
survey of 35 transplant centers found that only 65% of centers perform protocol liver biop

http://dx.doi.org/10.5772/52617
sies for HCV patients, and only 25% of centers perform protocol biopsies for other posttransplant patients [13].
The rationale for protocol biopsies is the detection of those patients with severe dysfunction
in the hopes that early treatment and/or change in immunosuppression might improve graft
survival. However, the evidence of the clinical utility of these biopsies is conflicted. In stud
ies of long-term protocol biopsies in non-viral hepatitis transplant patients, it does appear
that histological abnormalities in the setting of normal liver enzymes likely are not clinically
significant [49,50]. The rationale for the use of protocol liver biopsies in HCV patients is the
identification of those with severe HCV recurrence in the hopes that prompt treatment
could improve graft survival [51]. This appears to be justified, as several studies have dem
onstrated the clinical utility of protocol biopsies in HCV patients, even as long as 20 years
post-transplant [52-54]. It is notable, however, that the vast majority of patients with recur
rent HCV (and all patients with severe recurrent HCV) had abnormal liver enzymes at the
pre-determined time of protocol biopsy.
A separate but equally important factor in long-term patient survival is the avoidance of ex
trahepatic complications of chronic immunosuppression, including renal insufficiency, the
development of diabetes mellitus, and infectious complications. In this regard, another utili
ty of protocol liver biopsy is the identification of those patients in whom immunosuppres
sion can be safely lowered. A retrospective study of patients with various liver diseases
found that protocol biopsy results led to a change in immunosuppression in almost on third
of patients [55]. Recently, an international working group developed recommendations for
protocol biopsy monitoring in patients in whom minimizing or weaning immunosuppres
sion is being considered [56].
7. Summary
The liver allograft is susceptible to a broad range of insult and injury from the time that it is
removed from the donor. While some complications are easily diagnosed by the clinical pre
sentation and advanced imaging, the majority of conditions display overlapping clinical fea
tures. As the treatment of these various conditions can be radically different, a definitive
diagnosis is crucial. To that end, post-transplant liver biopsy continues to play a key role in
the evaluation of liver transplant patients with hepatic dysfunction. While the role of proto
col biopsies in patients with no biochemical evidence of hepatic dysfunction has begun to
fall out of favor (especially in non-HCV patients), the use of biopsy in immunosuppressionweaning protocols could promote a renewed interest in this methodology. The current data
support the use of protocol biopsies in HCV patients (particularly in the first few years posttransplant). Areas for future investigation include non-invasive alternatives to liver biopsy
such as immune assays and advanced imaging, and the use of routine protocol biopsies in
weaning of immunosuppression.
201
202
Author details
Alpna R. Limaye1, Lisa R. Dixon2 and Roberto J. Firpi1*
*Address all correspondence to: roberto.firpi@medicine.ufl.edu
1 Section of Hepatobiliary Diseases, Division of Gastroenterology, Hepatology, and Nutrition,
Department of Medicine, University of Florida, Gainesville, FL, USA
2 Department of Pathology, Immunology, and Laboratory Medicine, University of Florida,
Gainesville, FL, USA
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(OT). Liver Transpl. 2012 May 29. [Epub ahead of print].
Section 3
Non-Invasive Alternatives of Liver Biopsy
Chapter 10
Non-Invasive Evaluation of Liver Steatosis, Fibrosis and

Cirrhosis in Hepatitis C Virus Infected Patients Using
Unidimensional Transient Elastography (Fibroscan)
Monica Lupsor, Horia Stefanescu, Diana Feier and
Radu Badea
http://dx.doi.org/10.5772/52621
1. Introduction
1.1. The importance of non-invasive evaluation of liver steatosis and fibrosis in virus C
infected patients
Chronic conditions of the liver represent an important public health issue. Whatever the na
ture of the aggression against the liver, it seems that it always follows the same pattern: in
flamation -> necrosis -> healing (fibrosis) -> regeneration (cirrhosis) -> dysplasia ->
hepatocellular carcinoma. An important link in this course of events is represented by fibro
genesis. On the other hand, there are more and more evidence that, in patients with chronic
hepatitis C, steatosis is a risk factor independently associated with necroinflammatory activ
ity and fibrosis progression.
At the moment, the gold standard in the evaluation of both liver fibrosis and steatosis is rep
resented by liver biopsy (LB), an invasive method with possible side effects. As a result,
most of the research done worldwide is focused towards developing other alternative, noninvasive diagnosis methods, that would be capable to evaluate fibrosis and steatosis as accu
rately as possible.
Therefore, the following pages will present an evaluation of unidimensional transient elas
tography (TE) performance in the assessment of liver fibrosis and steatosis in patients suffer
ing from chronic viral hepatitis type C (HCV).
210
2. Liver biopsy An imperfect gold standard

Liver biopsy was performed by P Ehrlich in 1883 and became a common exploration meth
od in 1958, when G Menghini introduced the first biopsy technique with a needle that was
named after him [1].
Liver biopsy provides a lot of information [2-6]:
it represents the gold standard for a positive and differential diagnosis of diffuse liver diseases;
provides important etiology data;
allows for the evaluation of the necroinflammatory activity, evolutive stage (fibrosis) and
confirms if cirrhosis is present or not;
helps establish the prognosis;
identifies concomitent morphological alterations that may influence therapy response and
its evaluation (steatosis, iron overload, etc);
it can determine treatment efficiency.
At the same time, one can not ignore the fact that liver biopsy has significant limitations:
possible complications, including mortality; important sampling errors; high cost; subjective
appreciations that may be due to important intra and interobserver variations.
Biopsy complications may vary in magnitude and frequency depending on the subjacent liv
er pathology. Among these can be listed: pain (epigastric area, right shoulder, right hypo
condrium); vagal response; hemorrhagic accidents (hemoperitoneum, hemobilia, liver
hematoma); bile peritonitis, bilioma; bacteremia; infections and abscesses; pneumothorax
and/or pleural reactions; hemothorax; arteriovenous fistula; subcutaneous emphysema; ad
verse reactions caused by the anesthetic; breaking of the biopsy needle; penetration of other
organs: lung, kidney, colon. The mortality associated with this technique is low, but it is
possible in 0.0088-0.3% of the cases [7-11].
The most significant problem encountered when interpreting a biopsy is represented by
samplig error. Considering the fact that the tissue sample obtained through liver biopsy rep
resents approximately 1/50.000-1/100.000 of the liver volume, it can be inadequate for the di
agnosis of diffuse liver conditions, as the histopathological changes may be spread unevenly
[5]. Even though liver biopsy is considered the standard exploration in the evaluation of liv
er diseases, it has an accuracy of only 80% in staging fibrosis and it can miss cirrhosis in 30%
of the cases [12]. For example, Ragev reported that in HCV patients, there is a discrepancy of
at least 1 stage between the right and left lobe in 33% of the patients [13]. At the same time,
Siddique observed that a difference of at least one stage between 2 samples (15 mm long) cut
from the same area occurs in 45% of the cases [14].
Considering all these observations, the results of the studies performed to validate a noninvasive diagnosis method must be interpreted with caution, since they are compared with
an imperfect gold standard.
Non-Invasive Evaluation of Liver Steatosis, Fibrosis and Cirrhosis in Hepatitis C Virus Infected Patients Using
http://dx.doi.org/10.5772/52621
Because of the limitations and invasive nature of liver biopsy, other non-invasive means are
being tested for the evaluation of diffuse hepatopathies, and implicitly of fibrosis and steato
sis as major prognosis factors in the evolution of the hepatopathy. Therefore, there is interest
in developing other methods, either serological or imaging, which are all non-invasive, in
order to determine the presence and degree of fibrosis, as well as of steatosis. One of these
methods is unidimensional transient elastography (Fibroscan).
3. The principle of unidimentional transient elastography

The divice consists of a special transducer, that is placed in the axis of a mechanical vibrator.
The vibrator generates pain-free vibrations that produce a train of elastic waves that will be
transmited through the skin and subcutaneous tissue to the liver. At the same time with acti
vating the vibration, the probe performs a number of ultrasound acquisitions (the same
process of emision-reception used in conventional ultrasonography), with a frequency of 4
kHz. Reports on the tissues deformation caused by elastic wave transmission can be formu
lated by comparing the succesive ultrasound (US) signals acquired in this manner. The time
necessary for the train of waves to propagate along the area of interest, as well as propaga
tion velocities are being measured. This way liver stiffness can be determined using the fol
lowing formula: E = 3Vs2 (E elasticity module, density, a constant of the material; Vs
propagation velocity within the liver parenchyma). The more rigid the material, the higher
the velocity of propagation [15-17].
During the examination, the patient is lying down, face-up, with his right arm placed in hy
perextension and above the head for an adequate exposure of his right hypocondrium. The
probe is placed in contact with the patients skin, at the level of an intercostal space, in an
area of full liver dullness and avoiding any large vessels.
When the button on the probe is pushed, the vibration that will be transmitted through the
liver is activated. By analyzing tissue deformation report, the software of the equipment will
measure the liver stiffness (LS). The results are given in kiloPascals (kPa) and correspond to
a median value of 10 valid measurements. The machine can determine values between 2.5
and 75 kPa.
The monitor of the machine will display data regarding the patients identity, diagnosis,
name of the examining physician, the instantaneous value of liver stiffness (CS), the median
stiffness resulted from 10 valid measurements, the success rate (SR), as well as the variation
of the 10 values compared with the median value (IQR).
To be in agreement with the recommendations of the producer, the success rate must be at
least 60% and IQR must not exceed 30% of the median liver stiffness [16], even though it
seems that the best concordance with liver biopsy is obtained when this value does not ex
ceed 20% of the median [18].
There are no studies that especially focus on the issue of the variability of LS measurements
and therefore the interpretation of the results is done according to the experience of the ex
211
212
aminer and the recommandations of the producer [19]. It is not known whether this variabil
ity is encountered only in the diseased liver or whether it is present in the healthy liver as
well and to what degree this affects the interpretation of the results. The cause of this prob
lem can be an inadequate technique or the liver pathology itself (for example, in macronod
ular cirrhosis, liver stiffness can be different in different areas of the liver). When there is a
high variability of the results, it is important to check whether the probe is placed perfectly
perpendicular on the thoracic wall, if the transmited vibration does not encounter the ribs
and if the waves are transmited vertically, strictly between the ribs. If the generated wave is
large, bifid or angulated, than the software of the machine will reconstruct the velocity
curve in different points of the wave and therefore lead to variations of the acquired values.
In order to obtain an accurate elastogram the transducer must be placed in the middle area
of the right lobe, avoiding contact with the ribs that may lead to vibration distorsion and
absorbtion [19].
The technique measures the stiffness of a volume that is equivalent with that of a cilinder of
1 cm in diameter and 4 cm in length (the measurement can be performed on a distance of 25
to 45 cm from the skin). This volume, representing about 1/500 of the liver volume, is at least
a 100 times larger than the one obtained through liver biopsy and it is therefore more repre
sentative for the whole liver parenchyma [20, 21].
The examination can be performed by a technician following a short period of training (ap
proximately 100 cases) [22-23], while the clinical interpretation of the results must always be
done by an expert who would consider the demographic data, the etiology of the disease
and the biochemical profile of the patient at the moment of the examination [21].
A multivariate analysis of the relationship between liver stiffness and fibrosis, necroinflam
matory activity and steatosis Showed, in some studies, that there is a significant correlation
with fibrosis, but no correlation with necroinflammatory activity and steatosis [16, 24]. Nev
ertheless, the authors of the initial concept acknowledged, following in vitro studies, that it
is unlikely that a single physical parameter (liver stiffness) would describe entirely a com
plex biological system in which fibrosis is only a part [15].
A prospective assessment of the role of the histopathological parameters seen in LB in ex
plaining the variance of liver stiffness was performed on 345 chronic hepatitis C patients
that all underwent liver biopsy [25]. First, LS correlated highly with the degree of fibrosis
assessed by liver biopsy,, but we also found a weak correlation with hepatic iron deposition
and steatosis and a mild correlation with activity. In multiple regression analysis, fibrosis,
activity, and steatosis independently influenced LSM. Iron deposition does not seem to in
fluence the liver stiffness in CHC patients. Fibrosis, activity, and steatosis together explained
62.4% of the variance of the LS. The three significant parameters uniquely explained 45.95%
of the amount of LS, with fibrosis making the most unique contribution (44.49%); the differ
ence of 16.25% (62.4%-45.95%) was accounted for by the joint contribution of the three pa
rameters. The size and the direction of the relationships suggest that higher LS values are
obtained for patients with advanced fibrosis, increased necroinflammatory activity and in
creased steatosis. Among these three, however, the stage of fibrosis is the single most impor
tant predictor, as suggested by the squared partial correlation [25].
http://dx.doi.org/10.5772/52621
The prediction model computed from this study [25] can be expressed as follows:
Liver stiffness (log-transformed) = 0.493 + 0.180*fibrosis stage +0.034*steatosis + 0.033*activi
ty grade.
Therefore, our studies showed that fibrosis is indeed the main predictor of liver stiffness,
but the activity and steatosis cannot be neglected, and may explain the LS variability within
the same fibrosis stage.
4. Performance of TE for the noninvasive evaluation of liver fibrosis

HCV patients
The first condition that benefited from unidimensional transient elastography was chronic
hepatitis type C [21, 26].
4.1. The diagnosis of liver fibrosis stages
Studies performed on a large number of HCV patients indicate that the LS value is highly
correlated with the stage of fibrosis. The practical utility of the method is based on establish
ing cutoff values for each stage of fibrosis. A diagnosis of stage F 2, F 3 and F4 (cirrhosis)
is based on measurements of liver stiffness that vary, according to some studies, from 6.2 to
8.8 Kpa, 7.7 to 10.8 kPa and from 11 to 14.8 kPa (Table 1) [24, 26-30].
There are some meta-analyses addressing the issue of diagnosis performance of TE. Fifty
studies were included in the analysis performed by Friedrich Rust et al. The mean AUROC
for the diagnosis of significant fibrosis, severe fibrosis, and cirrhosis were 0.84, 0.89 and 0.94,
respectively [31]. In Stebbings meta-analysis, a total of 22 studies were selected, comprising
4430 patients, most of them suffering from a virus C liver infection. The pooled estimates for
significant fibrosis (F2) measured 7.71 kPa (LSM cutoff value) with a sensitivity of 71.9%
and a specificity of 82.4%, whereas for cirrhosis (F4) the results showed a cutoff of 15.08 kPa
with a sensitivity of 84.45% and a specificity of 94.69% [32].
It must be underlined that, in spite of the very good areas under the ROC curves, overlaps
of the stiffness values were registered in adjacent stages, especially for early fibrosis [33].
The increase of liver stiffness is higher between stage F2 (6.6 kPa) and F3 (10.3 kPa) of fibro
sis than between F1 (5.5 kPa) and F2 (6.6 kPa), a fact that is in agreement with the morpho
logical data according to which the increase in fibrotic tissue is more significant from F2 to
F3 than from F1 to F2 [12].
The diagnosis accuracy of TE is much better in predicting cirrhosis. In Friedrich-Rust metaa
nalysis [31], the AUROC mean for the diagnosis of cirrhosis was 0.94 and the performance
estimated by Talwalkar [34] was also very good: sensitivity 87%, specificity 91%, positive
probability rate 11.7, and negative probability rate 0.14 (95% CI 0.10-0.20).
213
214
Fibrosis
Stage
F1
F2
F3
F4
Author
Cutoff (kPa)
Se(%)
Sp(%)
PPV(%)
NPV(%)
+LR
-LR
AUROC
Ziol [24]
Castera [26]
Sporea [27]
Nitta [28]
Arena [29]
Kim SU [30]
Ziol [24]
8.8
56
91
56
88
0.63
0.48
0.79
Castera [26]
7.1
67
89
48
95
6.09
0.37
0.83
Sporea [27]
6.8
59.6
93.3
98
30.1
0.773
Nitta [28]
7.1
82.8
80.3
86
73.6
4.1
Arena [29]
7.8
83
82
83
79
4.58
0.20
0.91
Kim SU [30]
6.2
76
97.5
97.4
80
30.4
0.3
0.909
Ziol [24]
9.6
86
85
93
71
5.76
0.16
0.91
Castera [26]
9.5
73
91
81
87
8.11
0.29
0.90
Sporea [27]
Nitta [28]
9.6
87.7
82.4
72.5
92.7
0.90
Arena [29]
10.8
91
94
92
73
11.27
0.07
0.99
Kim SU [30]
7.7
100
95.7
87.5
100
23.3
0.993
Ziol [24]
14.6
86
96
97
78
23.05
0.14
0.97
Castera [26]
12.5
87
91
95
77
9.66
0.14
0.95
Sporea [27]
Nitta [28]
11.6
91.7
78
41.5
98.2
4.2
0.90
Arena [29]
14.8
94
92
73
98
11.27
0.07
0.98
Kim SU [30]
11
77.8
93.9
58.3
97.5
12.8
0.2
0.970
0.88
Table 1. Liver stiffness cutoff values for staging liver fibrosis using TE in HCV patients. Sensibility (Se), specificity (Sp),
positive predictive value (PPV) and negative predictive value (NPV) for each fibrosis stage (using Metavir scorring
system).
But it must not be forgotten that the cutoff values for predicting the stages of fibrosis were
chosen using the ROC curves in such a way that the sum of sensitivity and specificity is
maximum. The country where the study was performed was among the factors that influ
enced the diagnosis performance of TE [31]. Therefore, even though the cutoff values de
http://dx.doi.org/10.5772/52621
fined for a certain population may be relevant, they may not be applicable in another
population where the incidence of fibrosis is different. Because of this, it is indicated that
each centre establishes its own cutoff values, in agreement with the prevalence of fibrosis
stages in that particular population, and calculates the performance of the method in rela
tion with those cutoff values. According to our experience on a number of 1138 HCV pa
tients that underwent liver biopsy, the predictive cutoff values for stages F1, F2, F3 and F4
are: 5.1kPa, 7.5kPa, 9.1kPa and 13,2kPa, with an AUROC of 0.836, 0.826, 0.933 and 0.973, and
diagnosis accuracy between 77 and 92.8% [35]. In table 2 are presented the liver stiffness cut
off values that predict each stage of fibrosis for the Romanian patients suffering from viral C
chronic hepatitis. The table also presents the sensitivity (Se), specificity (Sp), positive predic
tive value (PPV) and negative predicting value (NPV), false positive (FPR) and false nega
tive rate (FNR) the area under the ROC curve (AUROC) as well as the diagnosis accuracy
(DA) of these cutoff values. In our study, the adjusted AUROC according to the prevalence
of each individual stage of fibrosis did not significantly differ from the observed ones (0.847
for F1, p=1.00; 0.893 for F2, p=0.06; 0.945 for F3, p=0.34; 0.983 for F4, p=0.312), therefore
the cutoff values that we obtained may have a large applicability.
4.2. Monitoring disease progression
4.2.1. Diagnosis of liver cirrhosis. Prediction of portal hypertension and related complications
TE has a very good diagnosis accuracy in predicting cirrhosis (stage F4 Metavir), with areas
under ROC varying from 0.90 to 0.99 and cutoff values between 9-26.6 kPa [31], but there is
a high interest to determine whether the use of the machines entire specter of measure
ments (up to 75 kPa) can predict the clinical events characteristic to the evolution of cirrho
sis. Some authors [36] indicated, with a negative predictive value of over 90%, that the
suggestive values for predicting the presence of various complications are: 27.5 kPa for large
esophageal varices; 37.5 kPa for Child B and C cirrhosis; 49.1 kPa for ascites; 53.7 kPa for
hepatocarcinoma and 62.7 kPa for bleeding esophageal varices.
Portal hypertension is the main characteristic of liver cirrhosis, and the hepatic venous por
tal gradient (HVPG) is the best surrogate marker to assess its presence.
A positive strong correlation between liver stiffness and HVPG was reported in HCV pa
tients [37] and, afterwards, independently confirmed in another group of patients with se
vere fibrosis (Metavir F3-F4) [38]. The correlation was excellent for HVPG values lower than
10 or 12 mm Hg, but the linear regression analysis did not reveal exceptional results for
HVPG values >10 mm Hg or >12 mm Hg. This means that, even though TU may detect a
progressive elevation of the portal pressure, mainly because of an increase in intrahepatic
vascular resistance caused by the accumulation of extracellular fibrillar matrix, this method
can not entirely determine the extremely complex hemodynamic alterations that character
ize the delayed phase of portal hypertension [39]. As a result, some authors believe it is un
likely that elastography can be useful in monitoring the hemodynamic therapeutic response,
as the effect of the treatment is mainly mediated by the splanchnic circulatory changes [40].
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F1; F0vsF1234
F2; F01vsF234
F3; F012vsF34
F4; F0123vsF4
Liver stiffness cutoff

value (kPa)
5.1
7.5
9.1
13.2
Se (%)
85.09
74.27
86.99
93.59
Sp (%)
65.45
82.95
88.51
92.71
+LR
2.46
4.36
7.57
12.84
-LR
0.23
0.31
0.15
0.07
PPV (%)
97.9
88.8
83.2
83.4
NPV (%)
18.7
63.9
91.2
97.4
FPR (%)
36.36
17.30
12.23
7.41
FNR (%)
13.86
25.59
12.55
6.41
Diagnosis accuracy (%)
85.01
77.34
87.63
92.87
Observed AUROC
0.836
0.860
0.933
0.973
0.847
0.893
0.945
0.983
1.00
0.06
0.34
0.312
Adjusted AUROC
according to the
prevalence of the
fibrosis stages
p (difference between
obs vs adj AUROC)
Table 2. Diagnostic performance of different cutoff values of liver stiffness in staging liver fibrosis in HCV Romanian
patients [35].
Regarding the relationship between liver stiffness and the presence of esophageal varices,
the area under the ROC curve for predicting the presence of varices varied between 0.76 and
0.84 [38, 41, 42]. Using cutoff values of 13.9 kPa, 17.6 kPa and 21.3 kPa, the sensitivity for
varices prediction was high (95%, 90% and 79%), but the specificity was relatively low (43%,
43% and 70%) [38, 41, 42]. There are studies that demonstrated a relationship between the
value of liver stiffness and the size of the varices [41, 42, 43], while other studies were not
able to demonstrate this correlation [38]. Using cutoff values of 19 and 30.5 kPa, the sensitiv
ity of TE for varices prediction was higher, but the specificity and the positive predictive
value were modest [41, 42]. TE did not provide better results than the serological markers
(like prothrombin time, thrombocytes [44] or FibroTest [45]), neither for varice detection (re
gardless of their grade), nor for the diagnosis of significant varices [41]. Yet, a predictive role
of liver stiffness in anticipating variceal bleeding cannot be excluded [43, 46].
These contradicting results may be caused by the heterogeneity of the studied populations,
the variable prevalence of varices (in general, but also of the large ones), the lack of prospec
tive validation (all the cited studies were cross-sectional studies) and the variability of the
cutoff values [47]. In conclusion, the evaluation of liver stiffness is not safe enough for the
detection and grading of esophageal varices in such a manner that it may replace upper di
gestive tract endoscopy in patients with cirrhosis, since the specificity and positive predic
tive value reported until now are too low to allow for a regular use of the method in clinical
practice.
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The literature data available on this topic are synthetized in table 3

Author
etiology
prev EV cutoff (kPa)

HVPG
VE
VEM
Carion[37]
HCV
8.7a
Bureau[43]
toate
21
Lemoine [48]
HCV
20.5
-OH
Vizutti [38]
HCV
66%
Kazemi [42]
toate
45%
Castera [41]
HCV
36%
Se
Sp
VPP
VPN
AUROC
90
81
90
81
0.92
90
93
91
90
0.94
63
70
35
88
0.76
34.9b
90
88
64
98
0.94
17.6
94
81
91
86
0.92
17.6
90
43
66
77
0.76
13.9
95
43
91
57
0.84
19
91
60
95
48
0.83
21.5
76
78
84
68
0.82
30.5
77
85
92
54
0.85
HCV = hepatits C virus; -OH = etanol; EV = esophageal varices; LEV =large esophageal varices; Se = sensibility; Sp =
specificity; P/NPV =positive/negative predictive value; AUROC = area under the ROC curve; HVPG = Hepatic venous
pressure gradient (a HVPG 6 mm Hg; b HVPG 10 mm Hg; c HVPG 12 mm Hg).
Table 3. TE performance in EV diagnosis and HVPG prediction in liver cirrhosis patiens.
The huge potential of TE for cirrhosis patients was acknowledged ever since the method
was introduced, as it can serve as a fast and non-invasive screening toll in the assessment of
actual complications, it can estimate the long term risk and thus place the patient in a certain
risk category [49]. The first signs of this possibility were the outcome of a retrospective
study which found that the risk of a patient with hepatitis C for developing hepatocellular
carcinoma is 5 times higher in patients with a LS value above 25 kPa, at the moment of the
diagnosis [50]. Even more, a recent prospective study [51], that evaluated the role of liver
stiffness in predicting complications related to portal hypertension in cirrhosis patients,
demonstrated that a LS value < 21.1 kPa at diagnosis was as valuable as a HVPG<12 mmHg
in the selection of the patients who will not experiment clinical events.
4.2.2. Optimization of liver stiffness performance in the diagnosis of liver cirrhosis or its
complications
Based on the principle enounced by Pinzani et al, which states that a concordance between
two distinct noninvasive tests is needed for an accurate diagnosis [52], an association be
tween LS and serum noninvasive tests for liver fibrosis was used to improve the diagnostic
accuracy. Such an algorithm was proposed by the Bordeaux group [53] and it is based on the
concordance between FibroScan and FibroTest. Using this approach, cirrhosis could be diag
nosed with an accuracy of 93% and liver biopsy could be avoided for the diagnosis of cirrho
sis in almost 80% of cases.
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On the other hand, our group managed to demonstrate that the Lok Score and LS used to
gether as part of a noninvasive algorithm (see figure 1) can improve (78% diagnostic accura
cy) the noninvasive estimation of large esophageal varices in cirrhotic patients [54].
Figure 1. Proposition for a non-invasive algorithm for the assessment of esophageal varices in patients with liver cir
rhosis.
4.2.3. TE efficacy in hepatocellular carcinoma risk assessment

Early detection of hepatocellular carcinoma (HCC) in HCV patients represents an emerging
health problem. As a common practice, alpha-fetoprotein (AFP) is widely used for the diag
nosis of HCC, despite its low sensitivity and specificity [55]. Tateyama et al report AFP
above normal levels as a risk factor for the development of hepatocellular carcinoma in pa
tients infected with hepatitis C virus [56].
Besides AFP, it has been proven that LS values increase as the liver disease progresses, the
highest values being specific for cirrhotic patients with associated HCC [36]. The evidence
prove that the individual role of increased LS measurements and serological markers are
predictive biomarkers of HCC [57, 58]. The first risk evaluation of HCC development in
HCV patients using TE was first performed by Foucher et al in 2006, reporting a cut-off val
ue of 53.7 kPa [36]. Also, according to Akima T et al [59] liver stiffness as measured by TE is
a good predictor of HCC development in viral hepatitis, with serum total bilirubin 1.0
mg/dL significantly correlated with tumor development. The latest published results report
good diagnostic accuracy of LS in HCC prediction, for cut-off values ranging between 12.5
and 53.7 kPa [57,59,60]. However none of these studies have been designed to evaluate the
accuracy of more predictive parameters, others than LS. On the other hand, adding bio
markers and other variables (such as variance) to LS measurement could represent con
founding factors in the assessment of patients with liver cirrhosis and HCC [61]. This may
lead to an over- or under-estimation of the risk assessed by TE, so a new accuracy testing is
http://dx.doi.org/10.5772/52621
needed. Nevertheless, increased LS seems to be a determinant of advanced cirrhosis, being

associated with decompensating episodes (high grade esophageal varices, bleeding, devel
opment of ascites) as well as with the presence of HCC [62], proving that increased LS alone
cannot be a good predictor of HCC.
On the other hand, Japanese studies also suggest that TE could be used as an indicator for the de
velopment of hepatocellular carcinoma in patients with virus C hepatitis [58, 63], the risk being 5
times higher in patients with a liver stiffness of over 25 kPa. Yet, these results must be confirmed
in prospective studies performed on larger groups of patients, in order to see whether liver stiff
ness can trully predict complication development in patients with compensated cirrhosis [21]. If
this fact is confirmed, elastography may serve as a non-invasive, quick screening modality which
could place the cirrhosis patient in a certain risk category [64].
4.2.4. Hepatitis C infection recurrence after liver transplant
ETU is useful in the appreciation of the severity of hepatitis C recurrence after transplanta
tion, thus reducing the number of liver biopsies [65]. In Carrion's study, for a cutoff value of
8.5 kPa, the sensitivity, specificity, negative predictive value and positive predictive value in
anticipating significant fibrosis were 90%, 81%, 79% and 92%. The important thing is that
none of the patients with a liver stiffness below that value presented severe fibrosis (F3), cir
rhosis (F4) or significant portal hypertension (HVPG 10 mm Hg). Furthermore only 6 (10%)
out of the 62 patients, having LS below the established threshold limit value,, did develop
portal hypertension, but in all cases it was a mild hypertension.
In Rigamonti's study [66], during the follow-up after transplantation, in 40 patients with
double biopsies (at 6 and 21 months), the liver stiffness changed in parallel with the stage of
fibrosis, having a sensitivity of 86 % and a specificity of 92% in predicting an increase in the
stage of fibrosis.
A recently published meta-analysis [67] showed that in patients undergoing transplantation for
HCV-related disease, TE appears to be a reliable diagnostic test for the exclusion of liver cirrho
sis. Furthermore, low TE values can reliable exclude cirrhosis in patients with recurrent HCV af
ter liver transplantation and liver biopsy may even be avoided in these situations. Among the
studies that evaluated significant fibrosis due to a recurrent HCV infection after liver transplan
tation, the pooled estimates were 83% for sensitivity, 83% for specificity, 4.95 for the positive like
lihood ratio, 0.17 for negative likelihood ratio and 30.5 for diagnostic odds ratio. For the studies
that assessed cirrhosis, the pooled estimates were 98% for sensitivity, 84% for specificity, 7 for
positive likelihood ratio, 0.06 for negative likelihood ratio, and 130 for diagnostic odds ratio[67].
5. Confounding factors influencing the interpretation of liver stiffness

values
Since the liver is self-contained in the non extensible Glissons capsule, stiffness is definitive
ly influenced by pressure that can be either hydrostatic or osmotic [68]. There are a few con
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ditions that may determine false results in situations where other factors, except from
fibrosis, are influencing liver stiffness.
Necroinflammatory activity proved to influence liver stiffness in patients with viral hepatitis,
causing an increase in stiffness in parallel with the grade of histological activity [29, 69, 70]. In
agreement with these results, the risk of overestimating the stage of fibrosis may occur in pa
tients with acute hepatitis or reactivated chronic hepatitis, if just the value of liver stiffness is con
sidered. Recent studies demonstrated that tissue alterations associated with acute hepatitis in a
patient with no liver disease history produce a significant growth of liver stiffness, sometimes
reaching cirrhosis values; this is due either to cellular intumescence or to severe cholestasis [71].
The contribution of these non-fibrotic changes upon liver stiffness was demonstrated by the pro
gressive reduction of liver stiffness parallel with the decrease of the transaminases [72, 73].
On the other hand, in patients with reactivated chronic hepatitis (therefore with preexisting
fibrosis), the increased stiffness is not caused by fibrosis alone, but also by the added cellular
intumescence [74].
From a practical perspective, it is important that the values of liver stiffness in patients with
acute hepatitis or in those with reactivated chronic hepatitis must be interpreted carefully,
within the patient's clinical and biochemical context [75]. In these patients, a certain diagno
sis of severe fibrosis or cirrhosis cannot be established. The right management in these cases
is to wait until the transaminases come back to normal and only when the potential involve
ment of inflammation is removed, the real status of fibrosis can be determined; it can thus be
established whether the event was an acute hepatitis on a diseased liver or a chronic hepati
tis with pre-existing fibrosis that was reactivated [76].
At the same time, in patients with acute hepatitis, the evaluation of liver stiffness at various
time intervals, can indicate the evolutive pattern of the condition, that may be characterized
either by evolution towards fulminant hepatitis (significant increase in LS), or by remission
(decrease in LS) [77].
Liver steatosis. The influence of steatosis on liver stiffness remains controversial. In some
studies, steatosis did not have a significant impact on liver stiffness, even after adjusting for
fibrosis stage [16, 24, 28]. Still, in these studies, the proportion of patients with severe steato
sis was too low to reliably quantify a possible influence and therefore further studies are
necessary to clarify this aspect.
We noticed from our experience that, after performing a stratified analysis of liver stiffness for
each stage of fibrosis, for the same grade of necroinflammatory activity (moderate-severe), the
presence of steatosis lead to a significant increase in LS from 5.89 1.64 kPa to 7.15 2.67 kPa for
those with stage F1 Metavir (p=0.004), and from 7.232.74 to 8.554.67 kPa for those with stage F2
(p=0.04) [78]. Besides, our studies have demonstrated that fibrosis is indeed the main predictor of
liver stiffness, but activity and steatosis cannot be neglected and may explain the LS variability
within the same fibrosis stage [25]. Afterwards, Ziol et al, using computer analysis of the micro
scopic image on a group of 152 patients, confirmed that steatosis clearly influences liver stiffness
independently from fibrosis, an influence that is insignificant in patients with cirrhosis, but im
portant in non-cirrhosis patients [79].
http://dx.doi.org/10.5772/52621
Extra-hepatic cholestasis. The impact of extrahepatic cholestasis on liver stiffness was re

cently demonstrated by Milloning [80] by evaluating cholestasis before endoscopic retro
grade cholangiopancreatography as well as 3 and 12 days after the procedure, in a study
group of patients with cholestasis caused mainly by a neoplastic invasion of the biliary
tree. If initially liver stiffness had values close to cirrhosis values (a mean of 15.2 kPa), af
ter drainage, the LS decreased as low as 7 kPa, in parallel with a decrease in values of bi
lirubin of 2.8-2.9 mg/dl. In all patients that underwent biliary drainage, the decrease of
liver stiffness correlated with that of the bilirubin values, with a mean of decrease of 1.2
0.56 kPa for a reduction of the bilirubin of 1g/dl. The relationship between liver stiff
ness and cholestasis was afterwards reproduced in the same study on an animal model
that underwent ligation of the biliary duct. This resulted in an elevation of liver stiffness
from 4.6 kPa to 8.8 kPa in the first 120 minutes after ligation and a decrease in stiffness
to 6.1 kPa within the first 30 minutes after decompression. In conclusion, it is indicated
that before an interpretation of the stiffness measurements is performed, an eventual ex
trahepatic cholestasis must be excluded using imaging investigations and lab tests.
Congestive heart failure may lead to an increased liver stiffness, with values similar to cirrho
sis, because of the elevated blood content of the liver, in 60% of the patients [80-84]. In the
context of cardiopulmonary conditions, TE may be relevant for the evaluation of treatment
efficacy, as liver stiffness decreases once cardiac compensation is achieved.
6. Optimizing the non-invasive diagnosis of portal hypertension using

spleen stiffness measurements
Splenomegaly is a common finding in liver cirrhosis that should determine changes in
spleen density as well, because of tissue hyperplasia and fibrosis [85, 86], and/or because of
portal and splenic congestion due to the splanchnic hyper-dynamic state [87]. These changes
might be quantified by elastography. Until recently, only magnetic resonance elastography
(MRE) was used with encouraging results in this respect [88]. The preliminary data showed
a highly signicant correlation between liver and spleen stiness in patients with portal hy
pertension, but, according to the authors, the validity of spleen stiffness as noninvasive
measure of portal venous pressure is not reliable enough [89].
6.1. Principle of TE for Spleen Stiffness Measurements (SSM) and technique assessment
Our group proposed for the first time the use of FibroScan for spleen stiffness measure
ment (SSM) [90]. For the measurement itself we proposed the same procedure as for the liv
er stiffness measurement, with the sole exception that the patient had his left arm in
maximum abduction and the transducer was placed in the left intercostal spaces, usually on
the posterior axillary line. For better locating the splenic parenchyma, we also used ultra
sound guidance, so that we could choose the best location for performing the analysis.
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6.2. Efficacy of spleen stiffness measurements for the evaluation of the presence and the
grade of esophageal varices
In the above mentioned study, we demonstrated that spleen stiffness can be assessed using
transient elastography, the sole factor influencing the measurement being the spleen size.
Spleen stiffness increases as the liver disease worsens, from normal to chronic hepatitis and
to liver cirrhosis (figure 2).
Figure 2. A - Box plots of spleen stiffness values for controls (0), chronic hepatitis (1) and cirrhosis patients (2). The top
and the bottom of the boxes are the first and third quartiles, respectively. The length of the box thus represents the
interquartile range within which 50% of the values were located. The line through the middle of each box represents
the median. The error shows the minimum and maximum values (range); B - Graphic representation of the significant
increase of SSM in healthy controls and patients with chronic hepatitis and liver cirrhosis, respectively.
In liver cirrhosis patients, the spleen stiffness measurement, can predict the presence, but
not the grade of esophageal varices. Therefore, for a cutoff value of 46.4 kPa, we managed to
predict the presence of esophageal varices with a diagnostic accuracy of 80.45% and an AU
ROC of 0.781 (figure 3).
Figure 3. A - Box plots showing the increase of SSM in liver cirrhosis patients with esophageal varices as compared
with those without; B - ROC curve representation of SSM in distinguishing LC patients with or without EV.
http://dx.doi.org/10.5772/52621
In another more recent study [91], another group demonstrated that SSM also correlates
with HVPG values, suggesting that this new elastographic technique may become a valua
ble noninvasive method for liver cirrhosis patients
6.3. Improving diagnostic accuracy for esophageal varices by modifying the SSM
calculation algorithm
Regarding the spleen stiffness measurement itself, we observed that the results seem to be
influenced by the intrinsic characteristics of the machine (FibroScan). Regardless of the vari
ceal status of the patients, or the grade of the varices, SSM reached the maximum value that
can be measured by the machine (75 KPa). This is an important drawback, because we have
to face a significant interpolation between the patients groups. If the FibroScan had been
able to determine values beyond 75 KPa, we may have obtained better figures. In order to
overcome this situation, we cooperated with the manufacturer of the device for developing
a new calculation algorithm, not available on the commercial device, which allows stiffness
measurements of up to 150 kPa. In a validation study [54], using the new calculation algo
rithm, we could differentiate between any classes of esophageal varices, except V1 vs V2
(p<0.005) and could select patients with V3 (V012 vs V3 = 63.49 vs 116.08 kPa, p<0.005), the
ones that are at higher risk for bleeding (figure 4).
7. Noninvasive evaluation of liver steatosis using Controlled Attenuation

Parameter (CAP)
Even though liver stiffness provides an alternative to liver biopsy for fibrosis staging, it can
identify very important histologic features such as macrovesicular steatosis, ballooned hepa
tocytes, inflammation, etc [68].
Figure 4. Boxplots representing mean SSM values according to the esophageal varices grade using the original (A) or
the modified (B) calculation algorithm.
Knowing that fat interferes with ultrasound propagation, a novel attenuation parameter has
been developed to detect and quantify liver steatosis. This parameter is based on the ultra
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sonic properties of the radio-frequency back propagated signals acquired by the Fibroscan
[92]. It is called controlled attenuation parameter (CAP). This ultrasonic attenuation coeffi
cient is an estimate of the total ultrasonic attenuation (go-and-return path) at the central fre
quency of the regular or M Fibroscan probe, i.e. at 3.5 MHz, and is expressed in dB.m1.
CAP is evaluated using the same radio-frequency data and the same region of interest, as
the region used to assess the LSM. CAP is only appraised if the acquisition is valid. There
fore, CAP is guided by vibration-controlled transient elastography (VCTE), which ensures
that the operator automatically obtains an ultrasonic attenuation value of the liver [92, 93].
The device is used to assess, at the same time, LS (which is related to liver fibrosis) and CAP
(which is related to liver steatosis).
Even though relatively few studies have been published on this topic [92, 93, 94,95] the pre
liminary results showed that CAP is a promising non-invasive tool to detect steatosis in
CHC patients.
In the study conducted by Sasso et al, the CAP performance was appraised on 115 patients,
taking the histological grade of steatosis as reference. CAP was significantly correlated to
steatosis with an AUROC equal to 0.91 and 0.95 for the detection of more than 10% and 33%
of steatosis, respectively.
A study performed recently on 615 HCV patients, who underwent both Fibroscan () and
liver biopsy showed in multivariate analysis, that CAP was related to steatosis, independ
ently of fibrosis stage (which was related to LS. The AUROCs of the were 0.80, 0.86 and
0.88 respectively, for predicting a fatty overload of more than 11%, 33%, and 66%, respec
tively. CAP also exhibited a good ability to differentiate steatosis grades (Obuchowski
measure = 0.92) [96].
CAP is evaluated using the same radio-frequency data and the same region of interest, as
the region used to assess the liver stiffness for fibrosis quantification. Preliminary studies
performed in our department have found significantly different CAP values for different
steatosis grades and AUROCs of 0.830 and 0.85 respectively, for the prediction of a hepatic
fat content over 33% and 66%, respectively [97].
8. The advantages of TE in liver steatosis, fibrosis and cirrhosis diagnosis

Compared with other diagnosis indicators or predictive models based on lab tests, the eval
uation of fibrosis using elastography has some important advantages [98,99]:
It is easy to use; noninvasive; pain-free; does not require anesthesia or hospitalization and
is therefore easily accepted by the patient;
It is quick, the time needed for the examination being very short;
It is not influenced by concomitant conditions;
It is operator independent;
http://dx.doi.org/10.5772/52621
The liver volume used to evaluate fibrosis is 150-400 times higher than the volume ob
tained through liver biopsy.
As far as the evaluation of liver steatosis is concerned, in comparison to other modalities,
CAP is non-invasive, quantitative, non-ionizing, and inexpensive. Furthermore, the proce
dure is easy to perform, even by an operator who does not have any radiological skills and
provides immediate results. The procedure is also machine-independent and does not re
quire corrections to be made for gain, frequency, focusing or beam diffraction, and is also
not subject to operator interpretation. In addition, CAP has been shown to efficiently detect
steatosis at a level of 10%, which is more sensitive than other imaging modalities. Com
pared to a liver biopsy, CAP is less prone to sampling error as it explores a liver volume
100 times larger [92, 93].
9. Limitations of TE
Liver fibrosis can not be evaluated by TE in 5-8 % of the cases. Some of the possible causes
for this are listed below [16]:
obesity (an ultrasound machine may be used in order to find the best window and thus
increase the ability to measure liver stiffness in overweight patients);
a narrow intercostal space;
ascites (vibrations are not transmitted through fluid);
the quality of the liver parenchyma and other liver structures;
large vascular structure present in the acquisition window (may lead to false results).
The failure of TE varies according to different authors from 2.4% to 9.4% [16, 21, 24, 36, 74,
64, 100]. In a study performed on 2114 patients [101], liver stiffness could not be determined
in 4.5% of the cases and multivariate analysis showed that the only element associated with
measurement failure is a body mass index over 28. Yet, with more experience, one may real
ize that a thick thoracic wall is more likely to be a limiting factor for a failed measurement
than the growth of the body mass index in itself [102].
Technical solutions regarding the design of the probe were investigated lately, in order to
overcome these limitations. Recently a new probe became available, that was specially de
signed for obese patients, with a central frequency of 2.5% MHz (compared with the 5MHz
probe that is usually used), and that is able to determine liver stiffness on a distance of 35-75
mm from the skin (while the normal probe is able to do that on distance of 25 to 45 cm).
With the help of this new transducer, it was possible to obtain valid measurements in 49% of
the patients with a BMI 30 kg/m2, in which the usual probe failed to determine the LS
[103].
As far as predicting steatosis in HCV patients is concerned, CAP has further validation in
larger populations and by independent teams, since there are rather few studies published
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until now. Another important limitation is that CAP cannot be used with measurements tak
en from the XL probe, which is a novel probe designed to assess liver stiffness in overweight
and obese patients [75,76]. Thus, CAP needs to be developed to work with the XL probe.
10. Conclusions
The possibility of concomitant assessment of liver fibrosis (using liver stiffness measure
ment) and of steatosis (using CAP) makes Fibroscan a promising non-invasive tool for as
sessing and quantifying both fibrosis and steatosis, that may broaden the spectrum of noninvasive methods used for the investigation and follow-up of patients with chronic hepatitis
C. But it is important that interpretation of the liver stiffness values be done by an experi
enced physician and always within the clinical and biochemical context of the patient.
Acknowledgments
This material is part of the research project no 27020/ 6/ 15.11.2011, entitled The non-inva
sive evaluation of fibrosis and steatosis in diffuse liver diseases by unidimensional transient
elastography Fibroscan from Iuliu-Hatieganu University of Medicine and Pharmacy,
Cluj-Napoca.
Author details
Monica Lupsor1, Horia Stefanescu2, Diana Feier1 and Radu Badea1
*Address all correspondence to: monica.lupsor@umfcluj.ro
1 Medical Imaging Department, Regional Institute of Gastroenterology and Hepatology Prof
Dr Octavian Fodor, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca,
Romania
2 Hepatology Department, Regional Institute of Gastroenterology and Hepatology Prof Dr
Octavian Fodor, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Ro
mania
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Chapter 11
Computer Image Analysis of Liver Biopsy Specimens in

Patients with Heroin Abuse and Coinfection
(Tuberculosis, HCV, HIV)
Ivan B. Tokin, Ivan I. Tokin and Galina F. Filimonova
http://dx.doi.org/10.5772/52971
1. Introduction
The computer morphometry in histopathology is n of the most perspective directions in
contemporary medicine including the hepatopathology. The potential advantages of meas
urement in histopathology have been recognized for many years [1]. The quantitative esti
mation has several advantages over conventional visual assessment such as objectivity and
reproducibility [2]. The employment of modern optical equipment and special computer
programs creates the possibilities for significant acceleration of quantitative analysis.
At present the computer morphometry has been rather intensively used to study liver
changes of the patients with chronic viral hepatitis. The quantitative assessment of the fibro
sis was performed mainly in chronic virus hepatitis C [3, 4, 5, 6, 7. 8, 9].
Many investigators considered that the quantitative evaluation of hepatic fibrosis was most
ly useful for assessing the origin, location and the stage of fibrosis. Using the morphometric
analysis is also very important for the correct evaluation of repeated biopsies [10]. Some in
vestigators studied the changes in liver fibrosis after the interferon therapy [11, 12, 13]. This
technique can be used in future for therapeutic trials by the estimation of the agents inhibit
ing the fibrosis progression [7].
Rates of fibrosis progression differ markedly in patients with HIV/HCV co-infection [14, 15,
16]. The natural history of hepatitis C virus infection in tuberculosis and in human immuno
deficiency virus-infected patients has never been studied with the use of the computer mor
phometric analysis of liver fibrosis progression. In this chapter the changes of liver biopsies
in patients with heroin abuse and infected by hepatitis C virus (HCV), human immunodefi
236
ciency virus (HIV), pulmonary tuberculosis (TB) were studied by the morphological and
computer morphometric analysis.
2. Patients and methods

2.1. Patients
13 male patients with co-infection of pulmonary tuberculosis (TB), chronic viral hepatitis C
(HCV) and human immunodeficiency virus (HIV) were investigated during the study. All
the patients used also the injections of heroin (Table 1).
The patients started their history as a rule from heroin using (mean duration 9.5 years) and
later all of them acquired HCV (mean duration 7.1 years), HIV (mean duration 4.7 years)
and TB at last (the duration of TB infection of the most part of patients was less than 1 year).
The diagnosis was established after careful examination of the patients: the anamneses of
diseases and life, laboratory analyses, virological and morphological studies. Serum level of
alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was expressed. The
upper limit of normal (ULN) of ALT was 41 U/L, AST 31 U/L.
2.2. Histological evaluation

To refine the diagnosis as well as for detection of the activity of pathological processes in the
liver, aspiration biopsy was taken from all the patients. All liver biopsies were performed to
the routine medical follow up program, using the standard Menghini procedure [17, 18].
Criteria for adequacy of the biopsy specimens included a core length of 10 mm and at least
5-6 portal tracts. So, only 9 biopsy specimens were used for further histological evaluation.
Four biopsy specimens were fragmented and werent used (Table 1). Samples were formal
in-fixed and paraffin-embedded. Serial paraffin sections were cut at 5 mcm. Hematoxylineosin and tolluidine blue stains were used.
Each biopsy for necro-inflammatory activity and fibrosis was assessed by two hepatologists.
Knodell Histology Activity Index (HAI) was used to grade histopathological lesions [19].
HAI was graded as minimal (scores 13), mild (scores 48), moderate (scores 912), or severe
hepatitis (scores 1318). METAVIR group scoring system was used for detecting the stage of
fibrosis [20]. Fibrosis was staged on the scale from F0 to F4, as follows: F0 = no fibrosis, F1 =
portal fibrosis without septa, F2 = few septa, F3 = numerous septa without cirrhosis, F4 = cir
rhosis. Only single patient showed any signs of cirrhosis. Fibrosis was also staged by Ishak
scoring system[21]. In the Ishak scoring system interface hepatitis (piecemeal necrosis), focal
necrosis in the lobule, portal inflammation were scored from 0 to 4, incomplete cirrhosis
(bridging necrosis with occasional nodules) and cirrhosis were scored from 5 to 6.
Computer Image Analysis of Liver Biopsy Specimens in Patients with Heroin Abuse and Coinfection ...
http://dx.doi.org/10.5772/52971
Patient
number
Biopsy
Sex
Age (years)
number in
next tables
Duration of infections (years)
Duration of
heroin
TB
HCV
abuse
HIV
(years)
male
26
male
26
fragment
11
11
11
male
27
male
31
male
31
fragment
male
32
fragment
10
17
male
33
12
13
15
male
33
male
34
18
unknown
10
male
34
11
11
male
36
fragment
13
13
12
male
37
10
13
male
39
16
16
Table 1. Characteristics of patients with heroin abuse and co-infection of TB, HCV, HIV. The patients are arranged
according to their age.
2.3. Computer digital analysis

Quantitative morphometric analysis was performed using an image analysis system consist
ing of a microscope (Leica DM 2500) with attached digital camera (Leica DFC 320 R2) and a
computer. Serial pictures of biopsy slices of patients with co-infection were photographed
by light microscope and were saved electronically. Serial microphotographs of biopsies
were made by an objective x20. The further process was performed with the computer pro
gram Adobe Photoshop CS 5.0. Serial microphotographs were mounted to receive the gener
al picture of liver biopsy (Figure 1). The digital image was converted into a binary image.
The two-dimensional patterns were measured by direct pixels counting on the binary im
ages under simultaneous visual control of the light microscopy.
Figure 1. General picture of the liver biopsy composed by computer microscopy (Obj. x20) using Adobe Photoshop CS
5.0. Total area of the biopsy is 11449177 pixels
Three main parameters were used for quantitative evaluation: the total area of portal zones,
the total area of intralobular infiltrates and necroses, as well as the total area of hepatic
237
238
vessels (central and sublobular veins). We considered the total amount of these main param
eters as non-parenchymal elements. Liver plates and sinusoids were attributed to the hep
atic parenchyma.
The measurement of portions (in percentages) of portal area, foci of intralobular necroses,
and vessels was estimated.
2.4. Statistical analysis
Statistical analysis was performed by tabulated processor Microsoft Excel 2003 and STA
TISTIKA 9.0.
3. Results
3.1. Features of histopathological structure of biopsies
Morphological analysis of liver biopsies of the patients heroin addicts with tuberculosis
(TB) and virus (HCV, HIV) co-infection showed that the extension of portal zones, the dam
age of limiting plates of liver cells and the formation of piecemeal and bridging necroses
took place practically in all biopsies (Figure 2).
Figure 2. Subfigure with two images. Section of the liver biopsy specimen of a patient with co-infection (TB, HCV, HIV)
and heroin abuse. Variants (a, b) of the development of interface hepatitis with piecemeal necrosis at the peripheral
zone of portal tract. Hematoxylin-eosin. Obj. x40
The peripheral regions of the portal zones were usually densely infiltrated by lymphocytes
and mononuclear histiocytes (Figure 2). Sometimes the lymphoid aggregates adjacent to the
damaged bile ducts were formed. Dense connective tissue elements developed more often
around the portal vessels (portal veins and hepatic arteries).
http://dx.doi.org/10.5772/52971
The appearance of focal lymphohistiocyte infiltrates and the formation of numerous intra
lobular necroses, containing hepatocytes, surrounded by lymphocytes (encircled hepato
cytes) were typical to peripheral and middle zones of liver lobules (Figure 3).
Figure 3. Subfigure with two images. Section of the liver biopsy specimen of a patient with co-infection (TB, HCV, HIV)
and heroin abuse. Variants (a, b) of the development of intralobular necroses containing encircled hepatocytes at the
middle part of liver lobule. Hematoxylin-eosin. Obj. x40
In the liver parenchyma the narrowing of sinusoids, as without of inflammatory infiltration

signs and with the elements of lymphohistiocyte infiltration and chains of lymphocytes in
side of them, was predominated (Figure 4).
There were features of moderate protein and vacuole dystrophia in all biopsies. In two cases
some hepatocytes contained large lipid inclusions (Figure 5). Disturbance of the lobular ar
chitecture was observed only in one biopsy (Figure 6).
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240
Figure 4. Section of the liver biopsy specimen of a patient with co-infection (TB, HCV, HIV) and heroin abuse. Severe
infiltration of intralobular sinusoids by lymphocytes and histiocytes at the peripheral zone of liver lobule. Hematoxylineosin. Obj. x20
Figure 5. Section of the liver biopsy specimen of a patient with co-infection (TB, HCV, HIV) and heroin abuse. Expan
sion and infiltration of portal areas, presence of intralobular necroses at the middle zone of liver lobules, deposition of
lipid droplets were in some hepatocytes. Hematoxylin-eosin. Obj. x10
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Figure 6. Section of the liver biopsy specimen of a patient with co-infection (TB, HCV, HIV) and heroin abuse. Strong
development of bridging fibroses and disturbance of the lobular architecture. Hematoxylin-eosin. Obj. x10
3.2. Quantitative image analysis for evaluation of pathological changes in liver biopsy
structure
Quantitative computer image morphometric analysis included three indexes. We calculated
separately the square (in pixels) occupied by portal zones, the square of intralobular focal
infiltrates and necroses and the square of hepatic vessels (central and sublobular veins).
The portal areas were divided into two groups: the portal zones with primary formation of
piece-meal necroses and the portal zones with primary formation of bridging necroses. We
took into account the calculation of portal zones fragments and septa. We also subdivided
the intralobular damages in liver in two groups: the focal lymphohistiocyte infiltrates with
out hepatocytes and the intralobular piecemeal necroses with encircled hepatocytes.
As for hepatic vein, we separately considered terminal hepatic veins (central veins) and sub
lobular veins. In each case we estimated the relative square of the above-mentioned indexes
in pixels and then calculated the specific parts in percents to the total square of biopsy.
We assigned the total sum of a specific part of portal zones, the specific part of intralobular
focal infiltrates and necroses and the specific part of the hepatic veins as non-parenchymal
elements. Respectively, the hepatic plates and sinusoids were remained in the composition
of the parenchyma.
Then we calculated the parenchyma indexes as the relation of non-parenchymal elements to
the parenchyma; these indexes characterized a certain degree of the replacement of the func
tioning hepatic tissue.
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242
The data obtained were summarized in the Tables 2, 4, 5, 6 and 7. The samples of biopsies
were arranged in sequence of increasing of non-parenchymal elements in bioptats.
The control group included the analysis of cohort of the patients with the monoinfection of
chronic virus hepatitis C (Table 3). The morphometric analysis of liver structure of the pa
tients belonging to given group was made earlier with the use of the method of the stereo
metric point morphometry [8].
3.2.1. The general characterization of morphometric data

The analysis showed that the specific part of non-parenchymal elements strongly varied in
the group of the patients with co-infection and heroin abuse: from 2.65% to 27.43% (Table 2).
Mean value of non-parenchymal elements was 12.082.38. The specific part of non-paren
chymal elements varied in cases of monoinfection of hepatitis C with the different degree of
activity from 2.16% 11.93%, the mean value was 6.940.77. Thus, the mean value of nonparenchymal elements of liver biopsy of the patients heroin addicts with co-infection ex
ceeded the mean value of non-parenchymal elements in liver biopsy of the patients with
monoinfection of hepatitis C (HV) in 1.74 times. The maximal value of the specific part of
non-parenchymal elements in case of co-infection was in 2.29 times higher than in cases of
monoinfection HCV (Tables 2 and 3).
Biopsy Parenchymal Non-parenchymal

number elements, %
elements, %
Ratio of non- Total area

parenchymal of portal
elements
zones, %
Total area
of spotty
infiltrates ,
%
Total area
of hepatic
veins, %
Chains of
lymphocytes
(absent or
present)
97,35
2,65
0,03
1,86
0,52
0,27
93,39
6,71
0,07
5,80
0,74
0,17
93,07
6,93
0,07
6,43
0,21
0,27
90,73
9,27
0,10
7,59
1,33
0,35
90,52
9,48
0,10
7,93
0,46
1,08
88,87
11,13
0,13
10,41
0,37
0,35
83,72
16,28
0,19
14,52
1,53
0,53
81,19
18,81
0,23
17,99
0,75
0,07
72,57
27,43
0,38
27,16
0,31
Table 2. Quantitative characteristics of liver biopsy specimens of patients with heroin abuse and co-infection of TB,
HCV and HIV by computer morphometric analysis
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Biopsy
ALT
numbe activity
r
(U/L)
Total area
Total
of
number
morphomet
of
ry (points microsco
of
pic
intersection fields(SU)
Parenchy
mal
elements,
%
Nonparenchy
mal
Chains of
Ratio of
non-
Total area
parenchy of portal
elements
mal
,%
elements
area, %
Total area
of spotty
infiltrates
,%
Total area
of hepatic
veins, %
lymphocy
tes
(absent
or
present)
s)
(x400)
95
97,84
2,16
0,02
1,79
0,05
0,32
15,
29450
20
18910
61
97,54
2,46
0,03
2,00
0,25
0,21
57
37690
126
96,49
3,51
0,04
2,30
0,46
0,75
14
17980
58
96,40
3,60
0,04
3,18
0,01
0,42
26
46190
149
95,70
4,30
0,04
3,15
0,12
1,02
104
70060
226
95,37
4,63
0,05
3,25
0,30
1,09
15
86800
280
95,30
4,70
0,05
2,91
0,02
1,77
42
37820
122
94,94
5,06
0,05
3,81
0,26
1,00
35
80290
259
94,82
5,18
0,05
3,93
0,84
0,41
10
441
89900
290
93,36
6,64
0,07
3,29
2,02
1,32
11
214
54560
176
91,33
8,67
0,09
7,24
0,89
0,55
12
187
70680
228
90,54
9,46
0,10
7,57
1,76
0,13
13
333
47720
152
90,32
9,68
0,11
7,51
1,17
1,00
14
107
32860
106
90,29
9,71
0,11
8,32
1,02
0,37
15
38
53514
193
89,44
10,56
0,12
6,23
2,61
1,71
16
122
49600
160
89,11
10,89
0,12
9,07
1,27
0,54
17
596
75330
243
88,24
11,76
0,13
9,26
1,56
0,94
18
162
60760
196
88,07
11,93
0,14
11,49
0,44
0,00
Table 3. Quantitative characteristics of liver biopsy specimens of the patients with monoinfection of chronic hepatitis
C by stereometric point morphometry
We made the comparative analysis of histopathological changes in liver biopsy structure in

the group of the patients heroin addicts and co-infected using the standard semi quantita
tive methods of the Ishak score evaluation (Table 4).
We determined the histological activity index HAI according to Knodell [19]. The stages of
the fibrosis development were defined by two ways: with the use of the research group
French METAVIR [20] recommendations and with Ishak method [21]. Under the METAVIR
system we evaluated the fibroses using five indexes where the maximal evaluation was 4
scores (F4 cirrhosis). It consisted 6 scores (F6 cirrhosis) according the Ishak system.
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244
Standard semi quantitative analysis methods for the most part of biopsies (6 patients from 9)
made possible to determine the same fibrosis stage: F3 according to the Ishak system and F2
according to the METAVIR system (Table 4).
Biopsy
number
Nonparenchymal
elements, %
HAI by score
Knodell
Stage of
fibrosis by
score Ishak
Stage of
fibrosis by
score
METAVIR
2,65
F3
F2
6,71
F3
F2
6,93
F3
F2
9,27
F3
F2
9,48
10
F3
F2
11,13
12
F3
F2
16,28
15
F4
F3
18,81
11
F3
F2
27,43
16
F5
F4
Table 4. Comparative characteristics of non-parenchymal elements specific parts, grading of histopathological lesions
(HAI) and the stages of fibrosis in liver biopsy specimens of the patients with heroin abuse and co-infection of TB, HCV
and HIV by computer morphometry and semi quantitative evaluation
At that time the quantitative computer image morphometric analysis showed (Table 2) that
among studied biopsies the specific parts of non-parenchymal elements differed significant
ly in various biopsies at the same fibrosis stages.
The minimal value of the specific part of non-parenchymal elements was 2.65%. These val
ues were 6.71% 6.93% (two biopsy specimens), 9.27% and 9.48% (two other specimens of
biopsy) and 11.13% (one biopsy specimen). Thus, in this case the methods of the semi quanti
tative score evaluation reflected only common regularities of the process of the fibrosis devel
opment. Meanwhile, the quantitative value of fibrosis was very essential for decision making
of the medical treatment tactic and the estimation of the medical treatment effectiveness.
The quantitative value of fibrosis is especially important in the process of repeated studies
for the determination of positive or negative dynamics of the fibrosis development. The his
tological activity index HAI according to Knodell proved to be more informative. HAI in
creased gradually from 8 to 16 points in accordance with the increasing of specific parts of
non-parenchymal elements in biopsies.
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3.2.2. Computer image analysis of portal zones

The majority of non-parenchymal elements were situated in portal zones. Therefore these in
dexes were analyzed in details (Table 5).
Biopsy
number
Total
Total area of
area of
portal zones
Portal area with piecemeal necroses Portal area with bridging necroses
nonparenchy
mal
Number
%
per
elements,
biopsy
Total
area, %
Number
per
biopsy
Minimal
Maximal
size, %
size, %
Total Number
Minimal Maximal
area, % per biopsy size, %
size, %
%
1
2,65
1,86
0,00
1,86
0,14
0,99
6,71
5,80
13
2,98
0,10
0,86
2,82
0,10
1,23
6,93
6,43
0,18
0,06
0,12
6,25
0,01
3,93
9,27
7,59
4,94
0,16
2,38
2,65
0,14
0,97
9,48
7,93
4,89
0,90
1,45
3,04
0,37
1,35
11,13
10,41
12
0,62
0,19
0,42
9,79
10
0,16
2,92
16,28
14,52
12
1,13
0,47
0,66
13,39
10
0,12
4,51
18,81
17,99
7,92
1,17
3,37
10,07
0,01
10,07
27,43
27,16
18
11,17
0,25
2,39
15,96
10
0,17
4,34
Table 5. Quantitative characteristics of portal zones in liver biopsy specimens of the patients with heroin abuse and
co-infection of TB, HCV and HIV by computer morphometric analysis
The amount of portal zones studied in each biopsy varied from 5 to 18. It depended on the
total biopsy volume. The mean value of the portal zones number was 9.8 91.34. The
amount of portal zones with piecemeal necroses varied from 2 to 8 (mean value was
3.440.72). The amount of portal zones with the septa and bridging necroses was more sig
nificant, it changed from 1 to 10 (mean value was 6.441.01).
In one case (biopsy specimen 8) the portal zone included several portal tracts forming the
extensive confluent bridging necrosis.Thus, the amount of portal zones with bridging ne
croses (6.44) exceeded in 1.87 times the amount of portal zones with piecemeal necroses (3.44).
The total specific part of portal zones varied from 1.86% to 27.16% (mean value was
11.082.42) (Table 5).
The specific part of portal zones with piecemeal necroses varied from 0.18% to 11.17%
(mean value was 3.761.21). The minimal size of such portal zones characterized mainly its
fragment, it changed from 0.06% to 0.9% (mean value is 0.370.13). The maximal sizes of
such portal zones characterized in general the degree of the portal zone extension, they
changed from 0.12% to 3.37% (mean value was 1.290.37).
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246
The specific part of the portal zones with bridging necroses (Table 5) changed from1.86% to
15.96% (mean value was 7.311.63). Thus, the specific part of portal zones with bridging ne
croses was practically in 2 times (1.94) more than the specific part of portal zones with piece
meal necroses.
The minimal size of portal zones with bridging necroses characterized in general the septa
fragments, it changed from 0.01% to 0.37% (mean value was 0.130.03). The maximal size
reflected more correctly the specific part of the portal zones with bridging necroses, it
changed from 0.97% to 10.07% (mean value was 3.370.91).
The quantitative computer image morphometric analysis showed that the significant exten
sion of portal zones with the destruction of the limiting plate and the development of piece
meal or bridging necroses took place in all bioptats of this patients group. In addition the
specific part of portal zones with bridging necroses exceeded considerably (in 1.9 times) the
specific part of portal zones with piecemeal necroses.
For comparison: the total specific part of portal zones changed from 1.79% t 11.49% (man
value was 5.350.68) at chronic hepatitis C monoinfection (Table 3).
Thus, the specific part of portal zones of liver biopsies of the patients heroine addicts with
tuberculosis and virus (HCV, HIV) co-infection was 2.07 times higher than the specific part
of portal zones of liver biopsies of the patients with the monoinfection HCV.
Moreover the bridging and piecemeal necroses were absent in the liver of the patients with
monoinfection HCV under minimal and low activity. Their appearance was noticed only if
the value of specific parts of non-parenchymal elements exceeded 4.7%.
We have not observed any difference between the amounts of piecemeal and bridging ne
croses in biopsy specimens with monoinfection HCV.
3.2.3. omputer image analysis of intralobular infiltrates and necroses
We analyzed the morphometric indexes of intralobular infiltrates and necroses (Table 6).
Intralobular necroses presented in all biopsies, their number varied from 6 to 38 (mean value
was 16.333.42). The amount of focal intralobular lymphohistiocyte infiltrates was signifi
cantly less in comparison with the intralobular necroses containing encircled hepatocytes.
The total number of focal intralobular infiltrates varied in different biopsies from 1 to 11
(man value was 3.671.09), whereas the total number of intralobular piecemeal necroses
varied from 4 to 28 (man value was 12.782.5).
The relation between piecemeal necroses and focal intralobular infiltrates was especially de
monstrative (Table 6). The number of piecemeal necroses in each biopsy was in several
times more (up to 10 times) than the number of focal necroses. The total number of intralob
ular piecemeal necroses was 115, whereas the number of focal intralobular infiltrates was
only 33, i.e. in 3.48 times less.
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Total
area of
Biopsy
number
nonparenc
hymal
elemen
ts, %
Total
Total number
area of
Focal lymphohistiocyte infiltrates
ular
Total
Total number
necrose necrose area ,% per

s, %
hepatocytes (piecemeal necroses)
of
intralob intralob
ular
Intralobular necroses with encircled
s per
Total
Minimal Maximal Total number Minimal Maximal
size, %
size, % area, %
biopsy
per
size, %
size, %
biopsy
biopsy
2,65
0,52
0,01
0,01
0,01
0,51
0,04
0,18
6,71
0,74
22
0,03
6,93
0,21
0,05
0,01
0,71
16
0,02
0,11
0,02
0,04
0,16
0,02
0,08
9,27
1,33
28
0,09
0,01
0,02
1,24
22
0,02
0,25
9,48
0,46
10
0,01
0,01
0,01
0,45
0,02
0,12
11,13
0,37
16
0,03
0,01
0,01
0,34
13
0,01
0,06
16,28
1,53
38
0,10
11
0,01
0,08
1,52
28
0,01
0,36
18,81
0,75
10
0,75
10
0,06
0,11
27,43
0,31
0,04
0,01
0,02
0,28
0,03
0,06
Table 6. Quantitative characteristics of intralobular necroses in the liver biopsy specimens of the patients with heroin
abuse and co-infection of TB, HCV and HIV by computer morphometric analysis
The total specific part of intralobular necroses varied from 0.21% to 1.53% (man value was
0.690.14). The specific part of the focal intralobular infiltrates varied from 0.01% to 0.1%
(man value was 0.040.01). The size of the minimal infiltrate was only 0.01%, the size of the
maximal infiltrate was 0.08% (man value was 0.020.01).
The total specific part of intralobular piecemeal necroses varied from 0.16% to 1.52% (man
value was 0.660.14). The minimal size of the specific part of intralobular piecemeal necroses
was 0.01% (man value was 0.030.01), whereas their maximal size was 0.36 % (man value
was 0.150.02).
The analysis of the total biopsy specimen (Figure 1) allowed attributing the topography of
the intralobular necroses distribution. Thus, under the middle degree of the parenchyma in
jury (HAI according to Knodell system up to 10 points) the small lymphohistiocyte infil
trates dominated in periportal zones of lobules. Under the high activity of the process (HAI
according to Knodell scoring system exceeded 10 points) the large piecemeal necroses domi
nated, they arranged mainly in the middle zones of lobules.
Hepatocytes surrounded by lymphocytes were well noticeable in large piecemeal necroses
(Figure 3b); it is perhaps connected with hepatocytes death, mediated by lymphocytes.
It is typically that the inflammatory infiltration of sinusoids and the formation chains of
lymphocytes in them are mostly expressed in large piecemeal necroses (Figure 4).
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248
So, the histological activity index HAI according to Knodell reached 15 points, the total
number of intralobular necroses reached 38 (28 from them were referred to piecemeal ne
croses) in the biopsy 7 (Tables 4 and 6). Remarkably that during the cirrhosis develop
ment (biopsy 9, fibrosis stage according to the METAVIR system scale was F4 cirrhosis)
the total number of intralobular necroses considerably reduced (6 piecemeal necroses and 3
focal infiltrates in one large biopsy; see Figure 6).
3.2.4. Computer image analysis of hepatic vessels
The amount of venous vessels in biopsy samples varied from 2 to 7 (mean value was
3.330.63). The central veins with endothelium which are often damaged predominated in
all biopsies (Table 7).
Total
Total
Terminal hepatic veins (central
area of Total number

Biopsy
number
non-
area of
of
parenc hepatic hepatic

hymal vessels , vessels
elemen
ts, %
Sublobular veins
veins)
per
Total
area ,%
Number
per
biopsy
Minimal
Maximal
Total
size , %
size, %
area, %
Number
per
biopsy
Minimal
Maximal
size, %
size, %
biopsy
2,65
0,27
0,27
0,09
0,11
6,71
0,17
0,17
0,03
0,14
6,93
0,27
0,27
0,03
0,18
9,27
0,35
0,35
0,07
0,11
9,48
1,08
0,13
0,02
0,05
0,95
0,95
0,95
11,13
0,35
0,35
0,01
0,11
16,28
0,53
0,53
0,04
0,31
18,81
0,07
0,07
0,03
0,04
27,43
Table 7. Quantitative characteristics of hepatic vessels in liver biopsy specimens of the patients with heroin abuse and
co-infection of TB, HCV and HIV by computer morphometric analysis
Sublobular veins were observed only in two biopsies, perhaps they did not get into biopsies
because of large sizes in comparison with central veins.
The total specific part of the hepatic vessels varied from 0.07% to 1.08% (mean value was
0.340.1). The specific part of the central veins varied from 0.17% t 0.35% (mean value was
0.180.04). The minimal size of the central vein was 0.01% (mean value was 0.030.01), the
maximal size was 0.18% (mean value was 0.080.02). The specific part of sublobular veins
reached 1.48%, maximal size 0.95%.
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On the whole it is possible to note the tendencies to the stable extension of vessels and the
damage of its internal walls. In addition, the sharp narrowing of intralobular sinusoids adja
cent to above mentioned vessels, took part in the contribution of the impairment of the proc
esses of the microcirculation inside of liver lobules. Perhaps the worsening of
microcirculation lead to the bypass ways of the circulation, this may be one of the reasons of
bridging necroses development.
3.3. Investigation of activity of alanin aminotransferase (ALT) and aspartate
aminotransferase (AST)
The measurement of liver enzyme activities (serum ALT and AST) are important for diagno
sis and assessment of liver diseases in clinical practice. However, ALT levels fluctuate in
chronic HCV infection and may fall into the normal range [22].The use of many medications
have been associated with elevated ALT levels [23]. In chronic hepatocellular injury, ALT in
creasing is more typical than AST. However, when the fibrosis progresses, ALT activity typ
ically declines, and the ratio of AST to ALT gradually increases [24], especially during the
development of cirrhosis [25,26].
We observed the increasing of the ALT and AST levels practically among all the patients
(Table 8).
Biopsy
Activity of
number ALT (U/L)
Activity of AST (U/L)
Ratio of
AST/ALT
90
48
0,53
36
32
0,88
45
42
0,93
140
90
0,64
162
179
1,10
48
39
0,81
90
68
0,75
88
93
1,05
106
84
0,79
Table 8. Activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in liver biopsy
specimens of the patients with heroin abuse and co-infection of TB, HCV and HIV
So, the ALT level changed from 36 to 162 points (mean value was 89.413.45). The AST level
varied from 32 to179 points (mean value was 7514.25). The AST/ALT ratio varied from 0.53
to 1.10 points (mean value was 0.830.06).
The mostly expressed increase of ALT and AST levels was discovered in the patients with
the samples of biopsy having the specific part of non-parenchymal elements up to 10% (Ta
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250
ble 8, samples of biopsies 4 and 5). As a rule the ferment activity rather reduced under
the fibrosis intensification. The AST/ALT ratio was increased in 3 patients. In other cases it
was closer to the upper border of the normal level.
We have not discovered any direct interconnections between the ferment activity levels and
the sizes of the specific parts of intralobular necroses. The intralobular piecemeal necroses
were dominant in this group of the patients; perhaps, the hepatocytes destruction was
caused by the special mechanism of the cell death (apoptosis).
4. Discussion
Detailed information about natural history of HIV/HCV co-infection is discussed in special
review article [27]. Some studies have suggested that human immunodeficiency infection
modifies the natural history of hepatitis C virus infection accelerating the progression of fib
rosis and the development of cirrhosis [28, 29, 30, 31].
Co-infection HCV/HIV is very often discovered among injecting drug users [32, 33]. Thus, it
was shown that about 90% drug users (consumers of heroin) are infected by hepatitis C vi
rus [34]. Intravenous heroin abuse induces significant morphological changes in liver tissue
(vesicular changes, fatty changes, chronic hepatitis, cirrhosis), and the severity of these
changes increases with years of heroin abuse [35]. Authors supposed that worsening of mor
phological changes in the liver happens mostly often because of a significantly reduced de
toxification functions of the liver.
Espinal, Perz, Baz, Hnriguez et al. [36] analyzed the clinical aspects of the co-infection
HIV and tuberculosis. Tuberculosis remains an important public health problem in the
world that has been exacerbated by HIV epidemic, resulting in increased morbidity and
mortality [37, 38]. The pathogenesis and mechanisms of inflammation and accelerated fibro
sis in co-infected patients are still poorly understood [28, 39].
At present investigation the peculiarities of patients with heroin abuse and co-infection (TB,
HCV and HIV) were analyzed (see Table 1). All the patients were males of the age from 26
to 39 years (mean value was 32.2 years). The heroin abuse was the longest (mean value was
13.6 years). Patients with HCV-infection occupied the second position of disease duration
(mean value was 7.1 years), than there were the patients with HIV-infection (mean value
was 4.7 years) and finally the patients with TB-infection (mean value was 3.5 years). At last
case the tuberculosis was discovered for the first time of 7 patients from 13 patients. It is
characteristic that Mycobacterium tuberculosis was not discovered in phlegm of any patients
under repeated analyses.
We could not detect any interconnections between the quantitative parameters of biopsy
specimen getting with the use of computer microscopy and for the duration of above-men
tioned observations.
Moreover the tendency to the diseases heaviness increasing is evident. The good example of
this tendency is the biopsy specimen 9: the duration of heroin abuse in this case com
http://dx.doi.org/10.5772/52971
posed 15 years, HIV 13 years, TB 12 years and HCV 9 years. In accordance with it the
cirrhosis developed in the liver of this patient (see Figure 6) and the segment of non-paren
chymal elements reached 27.43%. Among them the specific part of portal zones was preva
lent (27.16%).
The other peculiarity was the presence of the same stage of fibrosis (namely fibrosis F2 by
METAVIR scoring) and F3 (by Ishak scoring) in liver of the majority of the patients.
At that time the segment of non-parenchyma elements in liver of these patients varied from
2.65% to 11.13%, and the specific part of the portal zones changed from 1.86% to 10.41%. The
detailed information about discussion questions and interpretation of liver biopsy assess
ment by grading and staging systems was presented in recent works [40, 41].
The typical changes included the destruction of limiting plate, the expansion of portal areas
and the development of interface hepatitis, formation of short septa or bridging necroses.
The image analysis allows calculating of portal zones areas and intralobular infiltrates in dif
ferent fields of biopsy vision. The expansion of portal zones took place especially during the
development of interface hepatitis. As a rule, intensive lymphohistiocyte infiltration pre
dominates in such a type of portal zones.
The region of intralobular infiltrates strongly varies. Our investigation showed that intralob
ular infiltrates developed as a result of lymphocyte-mediated death of hepatocytes (apoptosis).
Earlier we studied the apoptosis in liver biopsy specimens of the patients with HCV with
the use of the TUNEL method [42, 43]. TUNEL-marked cells looked as small groups similar
to intralobular piecemeal necroses. All morphometric parameters were significantly higher
in comparison with monoinfection HCV [8].
5. Conclusion
Morphometric image analysis gives a possibility to evaluate quantitative parameters of nec
ro-inflammatory and fibrosis changes in liver biopsy of patients with mixed infections and
heroin abuse.
It is characteristic that the combination of different infections leads to the progression of liv
er inflammation and the increasing of the portion of non-parenchymal elements as a total
sum of portal areas, intralobular infiltrates and distended hepatic vessels.
The investigation showed significant intensification of necroinflammatory lesions. Lympho
histiocyte infiltration was typical both for portal zones and intralobular areas. These mor
phological indications could be connected with the change of the immune state of patients
as a result of combine effect of bacterial, viral infections and heroin abuse. So, numerous fac
tors have been associated with an increased risk of fibrosis progression in liver of such type
of patients.
251
252
Of course, it is necessary for more correct analysis to study the biopsies of the patients of
several control groups with the sequential cut-off of the definite factors. We plan to carry
out such investigation in future.
Quantitative analysis of digital images of total biopsies is indispensable to study the effec
tiveness of treatment tactics testing as the effect of therapy can be calculated as the percent
age of morphological changes in biopsy.
Author details
Ivan B. Tokin1*, Ivan I. Tokin2 and Galina F. Filimonova1,2*
*Address all correspondence to: ivan.tokin@rambler.ru
1 St.-Petersburg State University, Russia
2 North-Western State Medical University named after I.I.Mechnikov, Russia
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Chapter 12
Future Aspects of Liver Biopsy: From Reality to

Mathematical Basis of Virtual Microscopy
Ludmila Viksna, Ilze Strumfa, Boriss Strumfs,
Valda Zalcmane, Andrejs Ivanovs and
Valentina Sondore
http://dx.doi.org/10.5772/52753
1. Introduction
Tissue investigation remains one of the most reliable diagnostic ways in both general medical
practice and liver pathology. At present, the routine liver biopsy investigation should include
obtaining a representative tissue sample, adequate technological processing and application
of histochemical stain panel [1-5]. The evaluation must be done in accordance with up-to-date
disease classifications and validated diagnostic criteria [6]. Protocol approach is recommend
ed in order to decrease the variability in description. In case of chronic inflammatory liver dis
ease, semiquantitative evaluation of inflammatory activity by Knodell, Ishak, METAVIR or
Scheuer score, or analogous system [7-11] must be applied. Additional methods as immuno
histochemistry or polymerase chain reaction are applied by necessity. The morphological
evaluation of biopsy is a part of medical teamwork. It should be preceded by clinical and lab
oratory investigations and biopsy findings must be incorporated in the general patients in
formation. Many of these principles will remain in use in the nearest future. However, both
clinical diagnostics and medical research undergo almost unlimited progress. The upcoming
innovations in liver biopsy analysis include incorporation of digital image analysis, genetic
investigations and immunohistochemistry for functionally important molecules as cytokines,
cell cycle markers and viral life cycle markers into everyday practice.
2. Morphological evaluation of liver: Todays reality

Despite the fact that the histological assessment of liver tissues plays an essential role in the
diagnosis of liver diseases and the histological conclusion serves quite often as a basis for
258
establishing the diagnosis, there are factors or reasons to be taken into consideration which
can negatively influence the results obtained in morphological evaluation of liver biopsy.
We have "assessed" the percentage of each factors influence on the final result description
and conclusion regarding diagnosis, where "0" is considered a factor that does not affect the
evaluation and its outcome, but "100% the factor which actually hinders the correct diag
nosis of the disease. The factors that may affect the liver tissue morphological assessment
and diagnosis of disease are summarized in Table 1.
No.
Factors affecting liver tissue
Evaluation of impact on
morphologic assessment
final outcome in % *
1.
Biopsy site selection in liver tissues
100 0
2.
Fixation and transfer of biopsy sample
100 0
Comments
It can be completely nonrepresentative site, such as subcapsular

Chemical environment (composition)
and temperature of fixation can affect
the biopsy specimen
3.
4.
Selection of certain section out of the

whole sample
Selection and quality of staining (panel of
visualization methods) of the sample
50 0
If the whole sample is used, then the

error theoretically is not possible
If the sample is not stained for the
100 0
reason to label a certain substance,

e.g., Fe, the error can reach 100%
Thick or disrupted tissue sections can
5.
Quality of biopsy specimen sections or

microtomy
30 0
hinder the pathology from the

observer. The thickness should not
exceed 3-4 micrometres.
6.
Number of viewable visual fields
80 0
7.
Technical condition of the microscope
80 0
Inaccuracies can occur if only some

separate visual fields are examined
Incomplete quality of optical system
can hinder the pathology from the
observer.
If the specimen where the basic
pathology relates to fatty changes
8.
Selection of evaluation scale
100 0
(steatosis) is assessed according to

Knodell scale, then the assessment is
inadequate if compared to the actual
liver tissue damage
* 100% - affecting
0% - not affecting
Table 1. Factors affecting liver tissue morphologic assessment
Future Aspects of Liver Biopsy: From Reality to Mathematical Basis of Virtual Microscopy
http://dx.doi.org/10.5772/52753
Further we will have a look at each factor separately. The first reason which may significant
ly affect the final result is the incidental character of biopsy specimen collection by means of
blind biopsy. In case of diffuse liver damage, it is important to obtain liver specimen from
a representative site (which is not subcapsular) or under ultrasonographic (USG) control. If
the liver specimen is obtained during invasive procedure (laparoscopy or open abdominal
surgery), it is of high importance to give information about preferable biopsy site to the col
league obtaining liver specimen. The liver specimens obtained during surgery are certainly
more targeted. More or less qualitative methodological performance of tissue collection may
also cause certain imperfections affecting quality of specimen evaluation.
Presuming that the biopsy specimen is obtained from the site typical for the certain liver
pathology, one more important issue is the quality of biopsy specimens fixation and slicing.
The aspect of special tissue staining must also be looked at, because in case of absence of
examination request or list of preliminary diagnosis provided by clinician, that emphasizes
the need for particular staining, morphologist is unable to give an adequate diagnostic as
sessment of biopsy specimen. Thus diagnosis like haemochromatosis and other pathologies
known as "storage diseases" can be missed.
The next factor, i.e., selection of certain section out of the whole biopsy specimen, is an issue
arising only in case if the biopsy specimen is not examined throughout or along its horizon
tal length. The cross-sectioning gives the chance to analyze tissues on different "depths" or
levels of the biopsy specimen.
The technical condition or quality of the microscope and number of viewable visual fields are
to be considered seriously. Nowadays, the usual practice of the pathologist is a general over
view of the material to gain insight into overall picture, noticing the most typical and impor
tant peculiarities. Inaccuracies can occur if only some separate visual fields are examined.
The subjective component of the morphological assessment of liver specimens and interpre
tation of the observed changes and their compliance or adherence to one or the other pathol
ogy is essential also. The problem could be the qualification and experience of clinician to
put together or combine visual insight in the particular biopsy specimen and clinical diagno
sis made up of biochemical, immunological and genetic parameters, and to use the interpre
tation of morphologist properly for establishing the diagnosis.
Selection of morphological or histological evaluation scale is significant. These scales are
very advantageous for standardizing experts assessment, converting it into measurable
characteristics and helping the clinician to make final decision about patients diagnosis. In
case of light microscopy the issue of selection of evaluation scale is a factor with up to 100%
error probability. For example, the use of the Knodell scale for patient with steatohepatosis,
HAI = 0, leads to incorrect conclusion that the patient is healthy, especially if the biochemi
cal parameters of blood are not altered.
If in addition the electron microscopic investigation of sequential liver biopsy specimens are
done, obtained results and conclusions are also affected by the whole process of the above men
tioned biopsy specimen collection and processing. The electron microscopy is currently consid
259
260
ered as an auxiliary method or technique, yet in the age of high-tech medicine, processes
ongoing on the level of organelles are the ones which by characteristic ultrastructural changes
frequently refer to or indicate a particular pathology. The following must be strictly observed in
electron microscopy: 1) liver tissue sampling and slicing into 1 mm3 pieces without mechanical
ly squeezing them and immediate immersion in fixing solution; 2) chemical composition of fix
ing solution, temperature, sample fixation and rinsing time; 3) embedding of liver tissue
samples in mixture of epoxy resins in accordance with polymerization time of these resins; 4)
quality of sample cutting with ultramicrotome and contrasting with uranyl acetate and lead cit
rate; 5) all cells and their organelles visible in the ultra-thin slices under the electron microscope
have to be examined. It should be noted that resolution of transmission electron microscope
(TEM) is within the range of 0.2 to 2 nm and resolution of scanning electron microscope is 4 nm.
3. Virtual microscopy: The general principles

To reduce the potential inaccuracies in the processing and evaluation of biopsy specimens it
is important to look for modern solutions in order to maximize the efficiency of use of biop
sy specimens. One of the solutions could be application of virtual microscopy having exten
sive mathematical basis with fractal and entropy considerations as well as technological
support by appropriate software and hardware. Implementation of innovations into practice
could significantly increase the effectiveness of liver biopsy specimens.
The digital image analysis [12-14] and computed morphometry in general is considered an
important tool in pathology. It can decrease the workload of voluminous repeated measure
ments and increase the accuracy and objectiveness of the results. In several fields, e.g., im
munohistochemical and molecular typing of breast cancer, the application of digital image
analysis is already highly practical [13].
In virtual microscopy, the demands for mathematical basis are higher than in routine histol
ogy. This is illustrated by examples of entropy considerations, Delaunays triangulation or
fractal geometry and general non-Euclidean geometry for irregularly shaped biological ob
jects [14-16]. Sophisticated software must be elaborated as well. Additional technical re
quirements exist for image resolution and size, fast wide-band data transfer as well as
digital data storing [12, 13]. The slide scanners and visualisation software are available and
improve continuously [12].
Computed morphometry becomes more practical in association with virtual microscopy and
digital image analysis as well. As postulated in reference [16] the natural development of
science occurs from the ability to recognize, name and classify the object (corresponding to
the diagnosis, e.g., chronic viral hepatitis C) to semiquantitative, ordering measurements
(e.g., the activity assessment by Knodell or any analogous scale), finally reaching quantita
tive characteristics. Descriptive diagnoses and semiquantitative estimates are widely used in
the classic liver pathology. In order to gain sufficient reliability and fastness, scalar meas
urements would require digital assessment [16]. Computed morphometry on the basis of
virtual microscopy is a way towards scalar measurements.
http://dx.doi.org/10.5772/52753
The virtual microscopy can be performed in two different ways. Interactive virtual micro
scopy by whole slide imaging leaves the conclusion in the hands of pathologist. It changes
significantly the working tools from optical microscopy and subjective decisions to comput
er screen and objective measurements. The automated virtual microscopy is even more ex
citing as computer system should evaluate the diagnoses [14].
In liver pathology, the software develops regarding assessment of steatosis [17-19] and fib
rosis [20-24]. Necroinflammatory changes can be quantified as well [16].
Regarding liver ultrastructure, morphometric evaluation of hepatocyte volume can have
prognostic significance predicting survival as shown in liver cirrhosis associated with portal
hypertension [25]. Morphometric analysis of liver parenchyma in different alcohol-related
pathologic conditions has been tested with good results [26]. Thus, changes in the volume
fraction of parenchymal interstitial space and in the surface density of hepatocyte plasma
membrane, rough endoplasmic reticulum and outer mitochondrial membrane can be of im
portance for distinguishing between cirrhosis and non-cirrhotic states. Hepatocyte nuclear
volume fraction measurement can predict the survival in case of cirrhosis. Interestingly, few
images are necessary to perform these measurements thus helping to characterise even
scarce tissue material [26].
Combination of multiplex quantum dot immunostaining with high resolution whole-slide
digital imaging and automated image analysis has been described [27].
At present, the two most frequently studied targets for computer-assisted and/or digital im
age analysis in liver biopsies include steatosis and fibrosis.
4. Digital assessment of liver steatosis

Among Western population, liver steatosis is a frequent finding [28-29] as it is associated
with such common factors as chronic viral hepatitis [19], alcohol drinking, diabetes mellitus
or obesity [17]. It has been considered a risk factor for liver fibrosis [18, 19]. Steatosis, includ
ing non-alcoholic steatohepatitis [19] has become an important target in diagnostics and sci
entific research therefore highly reproducible measurements are necessary to evaluate the
course of disease, outcome and effect of treatment. The biopsy is still considered a gold
standard in the diagnosis and assessment of steatosis as the imaging including ultrasonogra
phy, computed tomography and magnetic resonance imaging can be affected by lower sen
sitivity [17, 30]. The severity of steatosis in liver biopsies can be graded by several semiquantitative systems (Table 2) assessing the eyeballed proportion of affected cells [30-35].
The present semiquantitative estimates are subjective and limit the possibilities of statistic
analysis [18]. Numerical value, expressing the exact percentage of affected cells would be
more reliable if an adequate biopsy is analysed. Such measurement is possible, especially in
computer-assisted way, but it would require architecturally arranged count of nuclei and fat
vacuoles per biopsy. Thus, the measurement would be time-consuming and accordingly ex
pensive. On the other hand, steatosis is relatively easy target for digital quantification of the
261
262
general fat amount due to the regular shape and distinct colour of fat vacuoles [18, 19]. The
digital quantification of steatosis shows high reproducibility exceeding the quality of man
ual estimate [19]. Commercial software for image analysis has been recently employed and
novel automated procedures are under development [18]. The estimate is more reliable if
both morphological and chromatic operators are used in order to characterise lipid particles
[18]. The fat vacuole is optically and geometrically simple object optically empty after rou
tine processing and deparaffinisation, thus white and rounded. If colour only is used for
identification, however, the sinusoids, empty portal vessels and bile ducts [30] as well as
glycogen nuclei in hepatocytes might be undertaken as false positives (Figure 1). The round
ed shape of fat vacuole helps to exclude longitudinal or tangential sections of sinusoids,
blood vessels and portal bile ducts. In haematoxylin-eosin stained sections, the colour con
trast can be used to identify glycogen nuclei as in this case the optically empty space is sur
rounded by basophilic nuclear membrane in contrast to fat vacuole located in eosinophilic
cytoplasm. Thus, the conclusion at present is to include both chromatic, size and shape as
sessment [18, 30]. Manual check can improve the accuracy in case of perpendicular sections
of small vessels and fat cysts [30]. However, such control would increase the workload. The
benefits of objectiveness and numerical value of continuous variable still remain. More stud
ies would be necessary to determine how accurate the control must be for practical means;
theoretically the significant vascular changes in cirrhosis point towards the idea that accu
rate identification of fat vacuoles is a must to avoid non-random errors.
Grading
Mild: less than 30% hepatocytes involved
Reference
[31]
Moderate: 30-60% hepatocytes involved

Severe: more than 60% hepatocytes involved
Grade 0 (no or minimal steatosis): less than 5% hepatocytes involved
[32]
Grade 1: at least 5% but less than 25% hepatocytes involved

Grade 4: at least 75% hepatocytes involved
Estimating the percentage of affected hepatocytes in 5% bands
[30]
Grade 0: less than 1%
[34]
[35]

Grade 4: at least 67% hepatocytes involved
Grade 1: less than 33%
Grade 3: more than 66% hepatocytes involved
Table 2. The different grading systems of liver steatosis
[33]
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Figure 1. Liver steatosis. Note the macrovesicular steatosis (stars) characterised by size of fat vacuole exceeding the
diameter of hepatocyte nucleus, and the microvesicular steatosis (small arrows) caused by fat vacuoles smaller than
hepatocyte nucleus. The optically empty fat vacuoles must be promptly distinguished from glycogen nuclei (large ar
row) and sinusoids (arrowhead). Haematoxylin-eosin stain, original magnification 400x
The fat stains as Sudan IV are well-known [4]. However, several researchers have reported tech
nical problems. The artifacts can include deformation of lipid vacuoles as well as sinusoidal and
background staining [17, 36-38]. The non-lipid positivity would limit the possibilities of colour
analysis, and the deformation of shape analysis. The practicality of osmium tetroxide stain is
negatively affected by the necessity to use frozen tissue and by the toxicity of reagents [4].
Several research groups have reported that manual assessment of steatosis leads to signifi
cantly higher estimates than computer-obtained data [17, 19] regardless if area measurement
or stereological point counting is used [30]. The coefficient can be as high as 3.78 [19]. Practi
cising physicians should remember that association between degree of steatosis and risk of
cirrhosis is proved using manual assessments and thus the scales are adjusted for manual
use. Consequently, interpretation of digital data cannot involve the use of unadjusted previ
ous scales as risk classes.
It should be noted that the principal meaning of diagnosing steatosis is not affected by the
evaluation method. Increasing steatosis percent is associated with advancing fibrosis stage
both manually and digitally [19]. The data obtained by pathologist and automated software
show close correlation [17]. After liver transplantation, aspartate aminotransferase, alanine
aminotransferase and prothrombin time have shown better correlation with automated
measurements in 4 of 5 posttransplant time points but the total bilirubin level correlated bet
ter with manual assessment in 3 of 5 time points. The graft survival showed a significant as
sociation with macrovesicular steatosis both in automated and manual measurements
although the p value was less for automated measurement [17].
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264
When analysing liver steatosis, the observations of higher accuracy in resin-embedded sam
ples [18] request more technological progress in order to create methodology for easy use in
routine samples.
Digital stereological point counting has been employed in liver steatosis evaluation as well
[33]. The researchers have observed the same fact that manual semiquantitative assessment
tends to be significantly higher. The lack of precision in manual evaluation can be related to
the physiology of vision and processing of the visual information [19, 39].
Some researchers have also come to the conclusion that automated assessment of liver stea
tosis is more time-consuming than manual [30]. The time input for digital measurement is
found to be threefold greater than for manual evaluation [19]. Although this opinion is
based on trustable experience, half of the problem is solved already as the whole slide imag
ing eliminates the need to choose appropriate number of representative fields submitted for
analysis and the necessity for human participation in the obtaining and archiving of digital
images. Besides the whole slide imaging, the degree of automatisation must be further in
creased: optimal software abolishes the manual correction of object inclusion into measure
ments. However, this deserves morphologically correct mathematical model. Other groups
have considered computer-aided morphometry to be fast and objective [16].
5. Digital assessment of inflammation in liver biopsy

The computer-aided assessment of necroinflammatory processes in chronic viral hepatitis has
been tested. To perform this, immunohistochemical visualisation is necessary in order to high
light inflammatory cells. The application of immunohistochemistry increases the expenses.
This drawback can be counterbalanced by gains of rapid measurement, resulting in rigorous
results expressed in scalar numbers as well as by complete characteristics more exactly reflect
ing the status of the whole organ [16].
The assessment of hepatic fibrosis and the closely related architectural deformities as bridging
fibrosis and liver cirrhosis have important role in the diagnostics, treatment and prognostic
evaluation of chronic liver diseases [24]. The studies of liver fibrosis are facilitated by standard
use of special stains for the routine evaluation of liver biopsies in case of diffuse liver disease.
Massons trichrome is an efficient method to highlight fibrosis [3]. The sharp contrast between
blue collagen and red parenchyma allows visualisation of even small excess amounts of colla
gen [23]. Sirius red stain has also been employed [21, 40]; it has the benefit of selective staining
of collagen but not proteoglycans [22]. Not surprisingly, comparatively many authors have ap
plied digital image analysis to quantify fibrosis in liver tissue [24]. Validation studies of com
puter-assisted morphometry have also been performed [21]. Besides the well-developed
methodology including software, the application of computer analysis has resulted in exact
numerical data allowing detection of interesting biological relationships. For instance, the cor
relation of fibrosis burden with end-stage liver disease score, serum total bilirubin and interna
tional standard ratio of prothrombin has been shown in hepatitis B-related decompensated
cirrhosis. Thus, the correlation between the amount of connective tissue in cirrhotic liver and
http://dx.doi.org/10.5772/52753
hepatic functional reserve was demonstrated [24]. The problem was insufficient accuracy of
computer-assisted morphometry [21] manifesting as inter-observer differences. Poor correla
tion of the fibrosis area with Ishak staging score has been observed as well [21]. Other scientists
have also found that analysis of early fibrosis necessities qualitative assessment despite the
general correlation between amount of connective tissue and Ishak grade of fibrosis [20]. Tis
sue geometry differences in subsequent sections also can be more accurately classified by hu
man eye [22]. Full section digital analysis seems to be important [20].
Digital image analysis for the evaluation of fibrosis in chronic viral hepatitis C has been
studied also as mentioned in references [41-42]. Automatic quantification of liver fibrosis in
cluding the validation of the method has been performed as described in reference [43]. Oth
er investigators have employed computerised image analysis for the evaluation of fibrosis as
well [44-47]. In most investigations, correlation between digital and manual semiquantita
tive score has been shown [20, 44-47]. However, the digital data do not allow to differentiate
between low stages of fibrosis [20, 45, 47].
6. Digital biopsy analysis for inflammatory liver lesion: Future begins

today
The incorporation of Mandelbrots fractal geometry [48] into the digital evaluation of liver
biopsy for chronic hepatitis has brought revolutionary changes [40].
The short description of fractal is provided in Table 3; detailed characteristics can be found
in recent reviews [49].
Definition and essential
The fractal is a mathematical object characterised by self-similar patterns. At every scale,
features of fractal
fractal shows (infinitely) either the same structure or is at least similar to other scales. The
complexity is retained independently of magnification. Thus, although fractal curve is one
dimensional similarly to regular line, the fractal dimension is greater than topological
dimension. Due to the infinite similarity, fractals cannot be measured in traditional ways.
Although fractals have got significant popularity due to their beauty, the importance of
fractal theory is in the mathematical basis and the ability to describe, among other
processes, the biological phenomena.
Fractals in nature: selected Beds of rivers, irregularity of coastline, profiles of mountain chain, clouds
examples
Fractals in biology: selected Branching of blood vessels or bronchi, the invasive edge of tumour, neurons. See also Figure 2-6
examples
Peculiarities of fractals in
Biological fractal-like objects have limited range of self-similarity upon magnification thus
biology
behaving as random fractals, in contrast to mathematical/geomaterical constructs with unlimited

level of complexity (self-exact fractals)
Table 3. The characteristics of fractals
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Figure 2. Highly irregular structure of biological object. Use of Mandelbrots fractal geometry is suggested to describe
targets with remarkable degree of complexity and irregularity. Note also the similarity of complex, branching outline
with Figures 4 and 5
Figure 3. Retained irregularity of the biological structure at higher magnification: note the remarkable similarity with
Figure 2. The persisting complexity at different levels of magnification is another feature suggesting the necessity for
fractal analysis. The inflammation in liver biopsy (shown in Figures 4 and 5) depicts analogous features
http://dx.doi.org/10.5772/52753
Hursts exponent is another albeit related mathematical construct with major meaning in the
digital analysis of liver biopsy. It was first used to study the variation in water flow in Nile ba
sin during the construction of the Aswan dam [16, 50]. In general, it can be used to detect the ir
regularity a key parameter analysing the activity of inflammation in the liver as the active
inflammation manifests with periportal piecemeal necrosis causing irregularity in the normal
ly smooth border of portal field. Hursts exponent also can be detected by fractal mathematics.
It can describe quantitatively the deviation from smooth contour in natural fractal objects.
To detect the border of inflammatory cell cluster, Delaunays triangulation can be used with
success. In general, Delaunays triangulation involves set of points in such way that no point is
inside the circle drawn through 3 points. It maximizes the minimum angle avoiding narrow
triangles. If circle drawn through 2 input points contain the third point in the outside, these
points form Delaunays triangle. The method can be used to mesh the space. By this triangula
tion, lines were drawn in the scanned image of liver biopsy through each pair of adjacent in
flammatory cells resulting in network of triangles showing common border. The most external
triangle short sides formed the border of inflammatory cell infiltrate. The triangle side was de
fined as appropriately short if it was equal of less than 20 microns based on empiric analysis.
After the cluster has been outlined, both the amount (by area) of inflammatory cells and the
border irregularity and area of cluster-affected tissue can be evaluated [16].
The mathematical basis of so-called geometry of irregularity (Figure 2-3) has allowed to detect
the amount of residual liver parenchyma, inflammation (Figure 4-5) and fibrosis (Figure 6-7) as
well as to provide index characterising the appropriateness of liver tissue structure (named tec
tonic index by the authors).
Figure 4. Irregular outline (arrowheads) of portal field in chronic active hepatitis. Haematoxylin-eosin stain, original
magnification 100x
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268
Figure 5. Branching pattern (arrowheads) of periportal inflammatory infiltrate. Note the remarkable similarity with
Figure 4 analogous to the relationship between Figures 2-3. The fractal nature of inflammation is thus highlighted.
Haematoxylin-eosin stain, original magnification 400x
Figure 6. Branching outline of connective tissue fields in liver cirrhosis. Note both the large areas of connective tissue
(star) and the thin septa (arrowheads). Massons trichrome stain, original magnification 100x
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Figure 7. Branching pattern of connective tissue fields in arachnoid liver fibrosis (arrowhead). Massons trichrome
stain, original magnification 400x
The Dioguardi Histological Metriser machine, described in reference [40] is able to pro
duce measurements and even simple diagnoses, working with reasonable speed. The rel
evant equipment ensures microscope focusing and full slide scanning, and determines
the above mentioned parameters excluding any unfilled spaces as vessels, sinusoids, bili
ary ducts and artifactual holes. The system is able to identify and exclude the Glissons
capsule from the analysis. Colour thresholds are used to select the areas of interest. The
inflammatory cells are identified by immunohistochemical visualisation of leukocyte
common antigen. For the analysis, the inflammatory cell clusters are outlined by imagi
nary line connecting the centres of the outermost cells; after that the area of clusters is
measured. Thinking in the usual terms, the portal and periportal infiltrates are character
ised by this measurement; the portal fibrosis also can influence this measurement provid
ing homing space for inflammatory infiltrate. The area of extra-cluster inflammatory cells
is measured separately; these could mostly correspond to intralobular infiltrate. When
analysing fibrosis, area of fibrotic tissue is measured. The wrinkledness is detected as the
ratio between the perimeter and area of an object. As portal field in healthy liver is
smooth, the concept of wrinkledness is an efficient way to detect periportal inflammation
and portal fibrosis. The irregularity of collagen islets necessitates the correction by fractal
dimension; the fibrotic foci are considered truncated planar fractals. The residual paren
chyma is characterised by the tissue area that is not occupied by inflammatory cells and
fibrosis. Finally, the loss of order is characterised mathematically. In order to characterise
the course of the disease in analogue with the usual staging, the individual fibrosis sca
lar is compared with the curve of fibrosis development over the course of disease detect
ing the percentage of the disease course before collagen deposition reaches the maximal
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270
tolerated level of 32% [40] or approximately 36% in liver cirrhosis necessitating liver
transplantation [24]. Thus, three approaches are combined: the outlines of regular struc
tures as vacuoles are characterised by traditional, non-fractal geometry, the area of fibro
sis and parenchyma are detected using the traditional measurements corrected by the
fractal dimension, and the tectonic index is based on the relationships between the Eucli
dean and fractal dimensions of liver tissue. One of the many positive features of this sys
tem is the ability to generate continuous scalar variables. When analysing dynamics in
repeated liver biopsies by scalar data, naturally, less biopsies are characterised as lacking
significant changes.
Although fractal concept is used in medicine, including at least microscopy, neuroscience
and ophthalmology as well as automated measurements not limited to pathology [49, 51,
52], the study described in reference [40] is remarkable as it is highly sophisticated and prac
tical; it is understandable that the research group considers their machine as an intelligent
collaborator and this is exactly the way how future biopsy analysis should proceed.
7. Functional liver tissue analysis in biopsy

The diagnostic evaluation of liver biopsy is mostly based on panel of histochemical
stains including hematoxylin-eosin [2], Massons trichrome [3], PAS [5] and Perls [1]
stains as well as others by necessity. These visualisation techniques should be comple
mented by various - omics tools [27] to gain more data on the function of liver cells.
The cytokines, inflammatory mediators, viral proteins, cell cycle proteins and apoptosis
markers can be detected; metabolic pathways can be investigated as well. At present,
most or proteomic and genetic studies are carried out for scientific research in order to
outline the pathogenesis of different diseases. However, in future it could be advisable to
include such studies with validated value in the routine investigation as technically the
amount of tissue in liver biopsy is sufficient.
Cytokine expression can be analysed, e.g., TGF, EGFR and others [25]. When studying inter
leukin-6 (IL-6) expression in liver biopsies, higher IL-6 expression was found in non-alcohol
ic steatohepatitis than in steatosis. Correlation between IL-6 expression and degree of
inflammation and stage of fibrosis was detected as well [53]. Due to the complex nature of
cytokine action, wide spectrum of different molecules and their receptors must be analysed
in details in order to avoid insignificant or contradictory results. This leads to a clear-cut ne
cessity for virtual microscopy and digital image analysis. Toll-like receptor-4 (TLR4) expres
sion can be analysed in liver biopsy by immunohistochemistry. The expression of TLR4 has
been shown in hepatic progenitor cells and interlobular bile duct epithelium in correlation
with stage of liver disease, grade of liver inflammation and activity of portal/septal myofi
broblasts [54]. The expression of interferon stimulated gene 15 can be analysed by IHC at
protein level; up-regulation in hepatocytes is more pronounced in patients not responding
to interferon / ribavirin treatment in contrast to predominant expression in Kuppfer cells in
treatment responders [55]. Proteomic studies including immunohistochemistry in liver biop
http://dx.doi.org/10.5772/52753
sy have targeted cell structure-associated proteins - actin, tropomyosin, transgelin and hu

man microfibril-associated protein 4 in order to identify biomarkers of liver cirrhosis [56].
COX-2 is over-expressed in chronic hepatitis C and the expression decreases following treat
ment with interferon alpha regardless of sustained virological response [57]. Increased en
doglin and TGF beta 1 expression is significantly associated with progressive hepatic
fibrosis in chronic viral hepatitis C [58].
Cell cycle analysis can add valuable information [59]; digital image analysis should be add
ed in the logistics again. Arrested cell cycle status has been demonstrated in chronic hepati
tis C infection analysing the expression of mini-chromosome maintenance protein-2 as
higher sensitivity proliferation marker, G1 phase marker cyclin D1, S phase marker cyclin A,
cell cycle regulators p21 and p53, apoptotic protein caspase 3 and anti-apoptotic protein
Bcl-2 [60, 61]. When analysing liver biopsies from patients with chronic viral hepatitis C,
higher G1 and lower S phase fractions has been found also by Werling et al., employing im
age analysis method [59]. Apoptosis-related pathways can be explorated including evalua
tion of Bax, Bcl-xL and Bcl-2 proteins [62]. Thus, hepatitis C virus infection can deregulate
the cellular processes [63] and it can be practical to reveal the way and degree of the regula
tory shift.
Viral antigens including hepatitis C antigen can be detected in liver tissue by immunohisto
chemistry [64]; the finding can be helpful in cases with difficult differential diagnosis or
combined liver pathology. The association of expression pattern with fibrosis may suggest
pathogenetically important information as well [64].
Metabolic pathways can be evaluated in liver biopsy. For instance, widespread expression of
vitamin D receptor has been shown in the hepatocytes and inflammatory cells in case of
chronic liver disease including non-alcoholic steatohepatitis and chronic viral hepatitis C.
The expression decreases as the liver histology is damaged [65].
Inflammatory cells are as important components in diffuse liver disease as the hepatocytes.
Thus, higher numbers of intrahepatic follicular T-helper lymphocytes in conjunction with
IL28B polymorphism analysis is found to be strongly predictive of treatment response using
pegylated interferon and ribavirin [66]. CD4+ regulatory T cells can be evaluated [67].
Logistic structures have been implemented to develop next generation toolkits for automat
ed image analysis to enable quantification of molecular markers. The group of researchers
[27] have collaborated within open source image analysis project [68] to reach effective out
put by combination of quantitative analysis, multiplex quantum dot (nanoparticle) staining
and high resolution whole slide imaging to detect nine different fluorescent signals for mul
tiple antigens [27].
DNA microarray technology has enabled genome-wide analysis of gene transcript levels.
This technology has been applied in order to compare gene expression profiles at different
stages of chronic hepatitis C and hepatocellular carcinoma in the setting of hepatitis virus C
infection [63, 69]. Hundreds of genes involved in carcinogenesis, cell growth, proliferation
and death are differently expressed in advanced viral hepatitis C in comparison to early vi
ral hepatitis C or non-viral hepatitis [63]. In chronic hepatitis C, the up-regulation involves
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272
genes related to metabolism and immune responses. In hepatocellular carcinoma arising in

hepatitis C patients, genes associated with cell cycle, growth, proliferation and apoptosis are
up-regulated [69]. Chronic hepatitis B and autoimmune liver disease have been studied by
this technology as well [70]. In advanced chronic viral hepatitis B, genes associated with ex
tracellular matrix turnover, cell growth and DNA repair are up-regulated but the expression
of genes regulating complement activation and innate immune response is decreased. In
early disease stages, the gene expression is different in case of chronic viral hepatitis B, auto
immune hepatitis and primary biliary cirrhosis. Chronic viral hepatitis B is associated with
expression of genes considering chemotaxis and cell homeostasis; autoimmune hepatitis
with down-regulation of genes associated with protein binding, but primary biliary cirrhosis
in early stages involves the actin and myosin gene expression. As chronic viral hepatitis B
progresses, the expression of genes regarding signalling pathway, cell communication, col
lagen turnover, chemokine ligands and metallothionein changes [70]. The findings are of
major interest displaying the pathogenesis of different inflammatory liver diseases and neo
plastic transformation. Diagnostic consequences should follow soon as the differential diag
nosis of inflammatory liver diseases regarding aetiology can represent a difficult task.
The level of mRNA can be post-transciptionally regulated by micro RNA (miRNA). The reg
ulation of biological processes by miRNA is shown also in case of such canonical diffuse liv
er disease as chronic viral hepatitis C. Technological studies have been conducted using
biopsy material [71]. Transcriptome analysis has shown prognostic value, e.g., in order to
predict the severity of fibrosis progression after liver transplantation in recurrent viral hepa
titis C patients [72].
8. Conclusions
Liver biopsy investigation could soon shift from routine light microscopy to digital image
analysis by virtual microscopy and incorporation of numerical measurements in conjunction
with integrated analysis of cell functions at DNA, RNA, protein and signalling level. This
shift could lead from static to dynamic tissue evaluation. The technological logistics should
include the best standards of tissue fixation, processing, microtomy and visualisation com
plemented by automated immunostaining, full slide scanning to ensure complete digital
analysis and optimal choice of software considering the biological appropriateness of the
analysis algorithm.
As the diagnostic electron microscopy is continually developing, we expect that in future it
will be used in hepatology as an auxiliary method, based on digital analysis of electrono
grams. Liver biopsy analysis using transmission and scanning electron microscope could
continue to provide important additional information in diagnostic hepatology and scientif
ic research of liver diseases, as well as it could help to study unresolved molecular mecha
nisms regulating liver cells functions. In future the ultrastructural studies of liver biopsy in
hepatology will probably be associated with assessment of liver tissues in cases of liver
transplantation, with studies of new medicinal products detection or exclusion of their po
http://dx.doi.org/10.5772/52753
tential hepatotoxic effect, with identification of viruses, as well as with determination of in

fluence of various environmental hazards.
Author details
Ludmila Viksna1,2, Ilze Strumfa1, Boriss Strumfs3, Valda Zalcmane1, Andrejs Ivanovs1 and
Valentina Sondore2
1 Riga Stradins University, Riga, Latvia
2 Riga Eastern Clinical University Hospital, Riga, Latvia
3 Latvian Institute of Organic Synthesis, Riga, Latvia
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Chapter 13
Real-Time Tissue Elastography and Transient

Elastography for Evaluation of Hepatic Fibrosis
Hiroyasu Morikawa
http://dx.doi.org/10.5772/52695
1. Introduction
Liver fibrosis develops as a sequel of chronic liver injury of various etiologies, including vi
ral infection, immunological reaction, and toxic and metabolic insults, and is characterized
by the accumulation of extracellular matrix(ECM) components produced by fibroblast-like
cells including activated stellate cells and myofibroblasts in the hepatic parenchyma. Hepat
ic fibrosis progresses towards cirrhosis, an end-stage liver injury, leading to hepatic failure,
hepatocellular carcinoma, and finally death. Hepatitis C virus (HCV) infection is the most
common cause of liver fibrosis. HCV infects approximately 170 million individuals world
wide according to a report from the
World Health Organization [1]. Liver biopsy has been considered the gold standard meth
od for the evaluation of liver fibrosis in chronic hepatitis C [2]. However, liverbiopsy has
some limitations, including its invasiveness, risk of complications, sampling error, variabili
ty in histopathological interpretation, and the reluctance of patients to subject to repeated
examinations [3-11].Because of these disadvantages, there is a growing shift inclinical prac
tice to utilize or develop non-invasivemethodologies to evaluate the stage of liver fibrosis.
In particular, liver stiffness measurement by Vibration-Controlled Transient Elastography
(Fibroscan) has become establishedas an important modality. Recently we and other investi
gator reported the usefulness of real-time tissue elastography (RTE) for noninvasive, visual
assessment of liver stiffness in patients with chronic hepatitis C [12.13]. RTE is a method in
tegrated in a sonography machine and developed in Japan for the visual assessment of tis
sue elasticity, based on a Combined Autocorrelation Method that calculates rapidly the
relative hardness of tissue from the degree of tissue distortion and which displays this infor
mation as a color image [14]. This technology has already been proved to be diagnostically
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valuable in the breast cancer [15]. We show here the additional value of RTE, in comparsion
to Fibroscanin patients with chronic liver disease.
2. Principle of elastography imaging

The two major categories of non-invasive hepatic elasticity imaging are dynamic elastogra
phy techniques, such as Fibroscan, and static elastography techniques, such as RTE. At
present the dynamic elastography techniques have the advantage of allowing a quantitative
imaging and better resolution than the static elastography techniques. These techniques re
quire more complex equipment for the generation mode and imaging modalities. Ultra
sound and magnetic resonance imaging are the major imaging modalities. The dynamic
elastography techniques may be devided into two groups, based on the method of generat
ing the shear wave: remote generation using radiation force and mechanical vibration. Of
the static elastography techniques, real time tissue elastography developed by Hitachi Medi
cal is most advanced ultrasound technique and can reveal tissue distortion using the hart
beat and pulsing of the aorta. Several elastography techniques are summarized in Table 1.
Principle
Mode of generation
Imaging modality
Real-time Tissue Elastography (RTE)
Tissue distortion
Pulsing of the aorta
Ultrasound
Vibration-Controlled Transient
Propagating shear wave
Mechanical vibration
Ultrasound
Radiation force
Ultrasound
Mechanical vibration
Magnetic resonance
Elastography (VCTE, Fibroscan)

Acoustic Radiation Force Impulse
(ARFI)
Magnetic Resonance Elastography
(MRE)
Supersonic Shear Imaging (SSI)
imaging
Radiation force
Ultrasound
Table 1. Elastography techniques for measurement of liver stiffness.
3. Real time tissue elastography

The principle underlining RTE is shown in Figure 1A, which illustrates this as a spring mod
el [16]. When a spring is compressed, the displacement in each section of the spring depends
on its stiffness: a soft spring compresses more than a hard spring. The strain distribution can
be measured by differentiating the spatial displacement at each location. Although the tissue
displacement usually is generated by manual compression and relaxation of the probe in
practice, we were able to improve the acquisition of RTE images representing the distortion
of liver tissue as a result of the beating of the heart or pulsing of the abdominal aorta.
Real-Time Tissue Elastography and Transient Elastography for Evaluation of Hepatic Fibrosis
http://dx.doi.org/10.5772/52695
RTE is carried out using a high quality ultrasound system (Hitachi AlokaMedical, Chiba, Ja
pan). The software uses a complex algorithm to process in a very short time all the data
coming from the lesion as radiofrequency impulses and to minimize the artifacts due to lat
eral dislocations, allowing accurate measurement of the degree of tissue distortion. We used
the Hitachi EUB-8500 and EUP-L52 Linear probe (37 MHz; Hitachi AlokaMedical) for RTE.
Figure 1. The principle and procedure of image analyses for real-time tissue elastography.(A) When a spring is com
pressed, displacement in each section of the spring depends on the stiffness of that part of the spring: a soft section
compresses more than a hard section. The strain distribution can be measured by differentiating the spatial displace
ment at each location. (B) The ROI was fixed to a rectangle of approximately 20-30 mm length x 20 mm breadth with
a 400600 mm2 area located 5-10 mm below the surface of the liver.left; RTE image, right; B-mode image. (C-D) The
color-coded images from the ROI of the RTE were analyzed by the software Elasto_ver1.5.1. The colors ranged from
blue to red indicating the relative gradients from hardness to softness. The Mean and Standard deviation were calcu
lated by a histogram, which was generated by 256 stepwise grading derived from the color image. The Area and Com
plexity were calculated from the binary image. Area was derived from the percentage of white regions (asterisks, i.e.
hard area). Complexity was calculated asperiphery2/Area. Median value of the data were recorded as representative
of RTE parameters.
This system is currently commercially available for the diagnosis of mammary neoplasm.
Patients were examined in a supine position with the right arm elevated above the head,
and were instructed to hold their breath. The examination was performed on the right lobe
of the liver through the intercostal space, and liver biopsy and Fibroscan also were per
formed at the same site. The RTE equipment displays two images simultaneously; one
shows the regions of interest (ROI) as a colored area and the other indicates the conventional
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B-mode image (Fig. 1B). We chose an area where the tissue was free from large vessels and
near the biopsy point. The measurement was xed to a rectangle 30 mm in length and 20
mm in breadth located 5-10 mm below the surface of the liver (Fig. 1B). The color in the ROI
was graded from blue (representing hard areas) to red (representing soft areas, Fig. 1B). We
stored the RTE images for 2- 3min as moving digital images (Fig. 1B) and ten static images
were captured at random from the moving images by the observer using AVI2JPG v6.10
converter software (Novo, Tokyo, Japan) and analyzed on a personal computer using the
novel software Elasto_ver 1.5.1,which was developed and donated by Hitachi Medical. Nu
merical values of pixels were from 0 to 255 (256 stepwise grading) according to color map
ping from blue (0) to red (255), and a histogram of the distribution was generated (Fig. 1C).
The scale ranged from red for components with the greatest strain (i.e., the softest compo
nents) to blue for those with no strain (i.e., the hardest components). Green indicated aver
age strain in the ROI, and therefore intact liver tissue was displayed as a diffuse
homogeneous green pattern. An appearance of unevenness in the color pattern was consid
ered to reflect a change in the liver stiffness. For quantification, all pixel data in the colored
image were transferred into a histogram and binary image (Fig. 1C, D).
4. Vibration-controlled transient elastography (Fibroscan)

Fibroscan, which has been developed for the measurement of liver stiffness, is currently con
sidered to reflect the degree of liver fibrosis directly and better than other methods. Fibro
Scan502 was developed by ECOSENS (Paris, France) to evaluate liver fibrosis noninvasively
in a short examination period by measuring the propagation of low frequency signals of a
mechanical shear wave running through the liver tissue. Fibroscan measures liver stiffness
in a volume that approximates a cylinder 10-mm wide and 40-mm in length between 25 and
65 mm below the skin surface. This volume is at least 100 times greater than that obtained
by liver biopsy and is therefore considered to be far more representative of the condition of
the hepatic parenchyma [17-21].The results that were obtained from ten valid measurements
with a success rate of at least 60% and an interquartile range under 30% were considered
successful. Failure was defined as when fewer than ten valid measurements were obtained.
The median of 10 valid measurements was expressed in kilopascals (kPa) and regarded as
the liver stiffness of a given subject.
Reports in 2005 from Castera et al. and Ziol et al. were pioneering; the liver stiffness measure
ments could be useful for assessing the presence of significant fibrosis (F2-4) and for suggest
ing the presence of cirrhosis in cohorts of patients with chronichepatitis C. The AUROCs
ranged from 0.79 to 0.83 for the prediction of F2-4 and were over 0.95 for the identification of
cirrhosis [22, 23]. Moreover, reproducibility of Fibroscan has been shown to be excellent for
both interobserver and intraobserver agreement with an intraclass correlation coefficient of
0.98 [24, 25]. Friedrich-Rust et al. [26] assessed the overall performance of TE for the diagnosis
of liver fibrosis by a meta-analysis that included fifty articles; the mean AUROCs for the diag
nosis of significant fibrosis, severe fibrosis, and cirrhosis were 0.84, 0.89, and 0.94, respective
ly. A recent report from Degos et al. [27] of amulticenter prospective study reported that the
http://dx.doi.org/10.5772/52695
AUROCs for the diagnosis of significant fibrosis and cirrhosis were 0.76 and 0.90, respectively.
Table 2 shows concisely the diagnostic accuracy of Fibroscan. The limitations of this method al
so have been discussed; intraobserver agreement is influenced by variables, such as body mass
index (particularly when<28 kg/m2), hepatic steatosis, and flares of transaminases [17.23].
Study
Patients (n)
Prognosis
Cutoff
Sen
Spe
PPV
NPV
AUC
(kPa)
Catera et al.
n=183, CHC
2005
Zioi et al.
n=251, CHC
2005
F2
7.1
67%
89%
95%
48%
0.83
Cirrhosis
12.5
87%
91%
77%
95%
0.95
F2
8.6
56%
91%
88%
56%
0.79
Cirrhosis
14.6
86%
96%
78%
97%
0.97
Friedrich-Rust et al. 50 studies, liver
F2
0.84
2008
disease
Cirrhosis
0.94
Degos et al. 2010
n=1307, viral
F2
5.2
90%
34%
64%
72%
0.76
hepatitis
Cirrhosis
12.9
70%
90%
53%
95%
0.79
Sen, Sensitivity; Spe, Specificity; PPV, Positive Predictive Value; NPV, Negative Predictive Value; AUC, Area Under the
Receiver-Operator-Characteristic curve; CHC, chronic hepatitis C.
Table 2. Diagnostic accuracies of transient elastography
5. Acoustic Radiation Force Impulse (ARFI) and Magnetic Resonance

Elastography (MRE)
The technology applied most recent is acoustic radiation force impulse (ARFI) imaging. AR
FI imaging permits evaluation of the elastic properties of a region of interest during realtime B-mode conventional hepatic US examination. Results are expressed in meters per
second and the region of interest can be chosen using ultrasound guidance, there by avoid
ing large blood vessels and the ribs. Previous reports have indicated that the diagnostic
power of ARFI imaging for the staging of liver fibrosis is the same as that of Fibroscan [28.
29].
New technological advances have been made in the clinical application of MRI such as dif
fusion-weighted MRI and MRI elastography. The former measures the apparent diffusion
coefficient of water and the parameter is dependent on the tissue structure [30]. The latter
measures the propagation characteristic of the shear waves from an acoustic driver within
the liver. Although MRI elastography has been shown to be superior to APRI and Fibroscan
for determining the stage of fibrosis in patients with various under lying liver diseases [31],
it cannot be performed on aniron-overloaded liver because of noise. In addition, MRI takes
longer and costs more than the ultrasound-base delastographic examinations.
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6. Our results
Patients: Two hundred and one patients with chronic hepatitis received liver biopsy and Fi
broscan examination within one week after RTE procedure in the Department of Hepatolo
gy, Osaka City University Hospital between 2007 and 2010. Etiologies of chronic liver
diseases were hepatitis C virus (CHC; n=129, 64.2 %), hepatitis B virus infection (n=13, 6.5
%), non-alcohol steatohepatitis (n=30, 14.9 %), and others (n=29, 14.4 %). Liver fibrosis was
evaluated according to the METAVIR score. Table 3 shows the characteristics of the patients
who received these examinations.
Sex: male/ female
89/112
Age
5513 y (21-80)*
BMI (kg/m2)
22.73.5 (14.1-33.2)*
Fibrosis stage(METAVIR Score)

F0
16
F1
98
F2
33
F3
27
F4
27
Etiology
HCV
129
NASH
30
HBV
13
Primary biliary cirrhosis
Others
14
BMI, body mass index.

Table 3. Characteristics of the patients
Results: Histological and laparoscopical examination: 16 (8 %) patients were classified as F0, 98

(49 %) as F1, 33 (16 %) as F2, 27 (13 %) as F3, and 27 (13 %) as F4 (cirrhosis). RTE was performed
successfully on all patients but Fibroscan measurements could not be obtained for 14 patients
(7.0 %)because of obesity and liver atrophy. The Mean decreased in proportion to the increase
of fibrosis score (JonckheereTerpstra test, p<0.0001). SD, Area, Complexity, and Fibroscan in
creased in proportion to the increase of fibrosis score (JonckheereTerpstra test, p< 0.0001).
http://dx.doi.org/10.5772/52695
Figure 2. Receiver operating characteristic curves of each parameter obtained by RTE and Fibroscan for F0-1.
Table 4 shows linear regression analysis of the values obtained by RTE compared to the liver
stiffness values obtained by Fibroscan. Although simple regression analyses indicated that
Mean, SD, Area, and Complexity were all significantly correlated with liver stiffness meas
ured by Fibroscan, the r value did not indicate a high correlation.
Mean
r=0.458
SD
r=0.377
Area
r=0.487
Complexity
r=0.451
p<0.001)
Table 4. Correlation between fibroscan and the image features of RTE
The area under the receiver operating characteristic curve (AUC) for stage F0-1 were 0.69,
0.65, 0.69, 0.67, and 0.87 for Mean, SD, Area, Complexity, and Fibroscan, respectively (Fig
2).The AUC for stage F0-2 were 0.79, 0.70, 0.77, 0.73, and 0.87 for Mean, SD, Area, Complexi
ty, and Fibroscan, respectively (Fig 3). The AUC for cirrhosis (F4) were 0.78, 0.68, 0.77, 0.76,
and 0.84 for each of respective values (Fig 4).
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Figure 3. Receiver operating characteristic curves of each parameter obtained by RTE and Fibroscan for F0-2.
Figure 4. Receiver operating characteristic curves of each parameter obtained by RTE and Fibroscan for F4.
http://dx.doi.org/10.5772/52695
7. Further research
Although our results showed that RTE was inferior to Fibroscan in determining the early
stage of liver fibrosis(Fig 2 and 3), Figure 4 indicated that the performance of RTE compares
favorably with that of Fibroscan for detecting liver cirrhosis in patients with chronic hepati
tis. Unfortunately the best method for the analysis and quantification of RTE remains un
clear, but this may be determined by future multicenter studies using larger patient cohorts
and the combination of these parameters will enable improvement of the accuracy of assess
ing hepatic fibrosis.
Fibroscan has been reported to have several limitations and disadvantages in evaluating pa
tients with obesity and ascites. In fact, in our study, we evaluated successfully all patients
with RTE, while Fibroscan measurements could not be obtained for fourteen patients be
cause of obesity and liver atrophy (data not shown).
In the future, a combination of imaging modalities and serological parameters or of different
imaging modalities will improve further the accuracy in differentiating fibrosis stages. Inter
estingly, Castera et al. reported that the best results were achieved by a combination of Fi
broscan and the Fibro Test [22]. Although ARFI, the most recent technology, Fibroscan, and
MRE are all based on shear wave propagation, RTE is constructed by an original theory
which is based on tissue distortion. The best diagnostic accuracy will be obtained by com
bining the RTE elasticity score with shear wave propagation.
8. Conclusion
We have described a static elastography technique, RTE, for the noninvasive visual assess
ment of liver stiffness. Although RTE was inferior to Fibroscan in determining the early
stage of liver fibrosis, the performance of RTE compares favorably with that of Fibroscan
when detecting liver cirrhosis in patients with chronic liver disease. We suggest that RTE
could also be used as a routine imaging method to evaluate the degree of liver fibrosis in
patients with other liver diseases. Future studies of larger patient cohorts will be necessary
for the validation of RTE analysis, and the combination of RTE with other clinical values in
cluding dynamic elastography techniques (i.e. Fibroscan, ARFI and MRE) and serum bio
markers will enable improvement of the accuracy of assessing hepatic fibrosis.
Acknowledgments
We thank Ms. Akiko Tonomura and Mr. Junji Warabino, Hitachi AlokaMedical Co., for the
technical support for RTE. Hiroyasu Morikawa was supported by a research grant from the
Cannon Foundation (2011-12).
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Author details
Hiroyasu Morikawa
Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka, Ja
pan
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LIVER BIOPSY INDICATIONS,

Liver Biopsy - Indications, Procedures, Results

Publishing Process Manager Viktorija Zgela

Liver Biopsy - Indications, Procedures, Results, Edited by Nobumi Tagaya

An Overview and Future Aspects of Liver Biopsy

Liver Biopsy - Indications, Procedures, Results 3

Types of Liver Biopsy 23

Rethinking the Role of Liver Biopsy in the Era

Clinical Practice of Liver Biopsy 73

Safety and Reliability Percutaneous Liver Biopsy Procedure in

Risks and Benefits of Liver Biopsy in Focal Liver Disease 85

Current Trends in Liver Biopsy Indications for

Primary and Metastatic Tumours of the Liver: Expanding Scope

Nonalcoholic Fatty Liver Disease: A Pathological View 161

Liver Biopsy After Liver Transplantation 189

Non-Invasive Alternatives of Liver Biopsy 207

Non-Invasive Evaluation of Liver Steatosis, Fibrosis and

Computer Image Analysis of Liver Biopsy Specimens in Patients

Future Aspects of Liver Biopsy: From Reality to Mathematical

Real-Time Tissue Elastography and Transient Elastography for

An Overview and Future Aspects of Liver Biopsy

Liver Biopsy Indications, Procedures, Results

Liver Biopsy - Indications, Procedures, Results

Liver Biopsy: An Overview

Liver Biopsy - Indications, Procedures, Results

Liver Biopsy: An Overview

Liver Biopsy - Indications, Procedures, Results

scopy. Electrocardiographic monitoring is required to detect arrhythmias induced by

Liver Biopsy: An Overview

Percutaneous LB with or without image guidance is appropriate only in cooperative pa

Liver Biopsy - Indications, Procedures, Results

Liver Biopsy: An Overview

Liver Biopsy - Indications, Procedures, Results

Liver Biopsy: An Overview

Neck hematoma, bleeding

Subclinical capsular perforation

Small hepatic hematoma

Transient Horners syndrome

Hepatic-portal vein fistula

Hepatic artery aneurysm

Inferior vena cava or renal vein perforation

Table 4. Complications of transjugular liver biopsy

Liver Biopsy - Indications, Procedures, Results

Liver Biopsy: An Overview

Liver Biopsy - Indications, Procedures, Results

Liver Biopsy: An Overview

Liver Biopsy - Indications, Procedures, Results

Liver Biopsy: An Overview

Liver Biopsy - Indications, Procedures, Results

Liver Biopsy: An Overview

[66] Chevallier P, Ruitort F, Denys A, et al. Influence of operator experience on perform

Liver Biopsy - Indications, Procedures, Results

Types of Liver Biopsy

2. Percutaneous Liver Biopsy (PLB)

Liver Biopsy Indications, Procedures, Results

3. Transjugular Liver Biopsy (TJLB)

Types of Liver Biopsy

4. Laparoscopic Liver Biopsy (LLB)

Liver Biopsy Indications, Procedures, Results

5. Transgastric Liver Biopsy (TGLB)

Types of Liver Biopsy

Liver Biopsy Indications, Procedures, Results

Figure 2. Hemostasis was confirmed at the site of liver biopsy.